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Marc Pansu 
Jacques Gautheyrou 
Handbook of Soil Analysis 

Mineralogical, Organic and Inorganic Methods 



Marc Pansu 
Jacques Gautheyrou 

Handbook 

of Soil Analysis 

Mineralogical, Organic 
and Inorganic Methods 

with 183 Figures and 84 Tables 



£}Spri 



ringer 



Dr Marc Pansu 
Centre IRD BP 64501 
Avenue Agropolis 911 
34394 Montpellier Cedex 5 
France 

E-mail : pansu@mpl.ird.fr 



Jacques Gautheyrou 
Avenue de Marinville 6 
94100 St. Maur des Fosses 
France 



Updated English version, corrected by Daphne Goodfellow. The original French book 
"L f analyse du sol, mineralogique et minerale" by Marc Pansu and Jacques Gautheyrou, 
was published in 2003 by Springer- Verlag , Berlin Heidelberg New York. 



Library of Congress Control Number: 2005938390 

ISBN-10 3-540-31210-2 Springer Berlin Heidelberg New York 
ISBN-13 978-3-540-31210-9 Springer Berlin Heidelberg New York 

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is 
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, 
broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication 
of this publication or parts thereof is permitted only under the provisions of the German Copyright 
Law of September 9, 1965, in its current version, and permission for use must always be obtained from 
Springer-Verlag. Violations are liable to prosecution under the German Copyright Law. 

Springer is a part of Springer Science+Business Media 

springer.com 

© Springer-Verlag Berlin Heidelberg 2006 

Printed in The Netherlands 

The use of general descriptive names, registered names, trademarks, etc. in this publication does not 
imply, even in the absence of a specific statement, that such names are exempt from the relevant 
protective laws and regulations and therefore free for general use. 

Cover design: E. Kirchner, Heidelberg 
Production: Almas Schimmel 
Typesetting: SPI Publisher Services 
Printing: Krips bv, Meppel 
Binding: Stiirtz AG, Wiirzburg 

Printed on acid-free paper 30/3141/as 543210 



Foreword 

This new book by Marc Pansu and Jacques Gautheyrou provides a 
synopsis of the analytical procedures for the physicochemical analysis of 
soils. It is written to conform to analytical standards and quality control. 
It focuses on mineralogical, organic and inorganic analyses, but also 
describes physical methods when these are a precondition for analysis. It 
will help a range of different users to choose the most appropriate method 
for the type of material and the particular problems they have to face. The 
compiled work is the product of the experience gained by the authors in 
the laboratories of the Institute of Research for Development (IRD) in 
France and in tropical countries, and includes an extensive review of the 
literature. The reference section at the end of each chapter lists source 
data from pioneer studies right up to current works, such as, proposals for 
structural models of humic molecules, and itself represents a valuable 
source of information. 

IRD soil scientists collected data on Mediterranean and tropical 
soils in the field from West and North Africa, Madagascar, Latin 
America, and South East Asia. Soil materials from these regions are often 
different from those found in temperate zones. As their analysis brought 
new problems to light, it was essential to develop powerful and specific 
physicochemical methods. Physicists, chemists and biologists joined 
forces with IRD soil scientists to contribute knowledge from their own 
disciplines thereby widening its scope considerably. This work is the fruit 
of these experiments as applied to complex systems, involving soils and 
the environment. 

The methodological range is particularly wide and each chapter 
presents both simple analyses and analyses that may require sophisticated 
equipment, as well as specific skills. It is aimed both at teams involved in 
practical field work and at researchers involved in fundamental and 
applied research. It describes the principles, the physical and chemical 
basis of each method, the corresponding analytical procedures, and the 
constraints and limits of each. The descriptions are practical, easy to 
understand and implement. Summary tables enable a rapid overview of 
the data. Complex techniques are explained under the heading 'Principle' 
and concrete examples of methods include: spectra (near and far IR, UV- 
visible, ! H-NMR, 13 C-NMR, ESR, ICP-AES, ICP-MS, X-ray 
fluorescence, EDX or WDX microprobe, neutron activation analysis), 
diffractograms (XRD, electron microdiffraction), thermograms (DTA, 
DTG, TGA), chromatograms (GPC, HPLC, ionic chromatography, 
exclusion chromatography), electrophoregrams, ion exchange methods, 
electrochemistry, biology, different physical separation techniques, 
selective dissolutions, and imagery. 



VI Foreword 

The book will be valuable not only for researchers, engineers, technicians 
and students in soil science, but also for agronomists and ecologists and 
others in related disciplines, such as, analytical physical chemistry, 
geology, climatology, civil engineering and industries associated with 
soil. It is a basic work whose goal is to contribute to the scientific 
analysis of the environment. The methodologies it describes apply to a 
wide range of bioclimatic zones: temperate, arid, subtropical and tropical. 
As with the previous books by the same authors (Pansu, Gautheyrou and 
Loyer, 1998, Masson, Paris, Milan, Barcelona; Pansu, Gautheyrou and 
Loyer, 2001, Balkema, Lisse, Abington, Exton, Tokyo), this new book 
represents a reference work for our laboratories. We are confident its 
originality and ease of use will ensure its success. 



Alain Aventurier, Director of Analytical Laboratories of CIRAD 1 
Christian Feller, Director of Research at IRD 2 
Pierre Bottner, Director of Research at CNRS 3 



CIRAD, Centre International pour la Recherche Agronomique etle 

Developpement (France). 
IRD, Institut de Recherche pourle Developpement (ex ORSTOM, France). 
CNRS, Centre National de la Recherche Scientifique (France). 



Contents 

Part 1 - Mineralogical analysis 

Chapter 1 Water Content and Loss on Ignition 

1.1 Introduction 3 

1.2 Water Content at 105°C (H 2 (T) 6 

1.2.1 Principle 6 

1.2.2 Materials 6 

1.2.3 Sample 6 

1.2.4 Procedure 7 

1.2.5 Remarks 7 

1.3 Loss on Ignition at 1,000°C (H 2 + ) 8 

1.3.1 Introduction 8 

1.3.2 Principle 11 

1.3.3 Equipment 11 

1.3.4 Procedure 11 

1.3.5 Calculations 12 

1.3.6 Remarks 12 

Bibliography 12 

Chapter 2 Particle Size Analysis 

2.1 Introduction 15 

2.1.1 Particle Size in Soil Science 15 

2.1.2 Principle 17 

2.1.3 Law of Sedimentation 18 

2.1.4 Conditions for Application of Stokes Law 24 

2.2 Standard Methods 26 

2.2.1 Pretreatment of the Sample 26 

2.2.2 Particle Suspension and Dispersion 31 

2.2.3 Pipette Method after Robinson-Kohn or Andreasen 35 

2.2.4 Density Method with Variable Depth 42 

2.2.5 Density Method with Constant Depth 47 

2.2.6 Particle Size Analysis of Sands Only 48 

2.3 Automated Equipment 50 

2.3.1 Introduction 50 

2.3.2 Method Using Sedimentation by Simple Gravity 51 

2.3.3 Methods Using Accelerated Sedimentation 53 

2.3.4 Methods Using Laser Scattering and Diffraction 54 

2.3.5 Methods Using Optical and Electric Properties 55 

2.3.6 Methods Allowing Direct Observations of the Particles 55 

2.3.7 Methods Using Conductivity 56 

References 56 

Bibliography 58 

Generality 58 



VIII Contents 

Pre-treatment 58 

Pipette Method 61 

Hydrometer Method 62 

I nstrumental Methods 62 

Chapter 3 Fractionation of the Colloidal Systems 

3.1 Introduction 65 

3.2 Fractionation by Continuous Centrifugation 66 

3.2.1 Principle 66 

3.2.2 Theory 69 

3.2.3 Equipment and reagents 73 

3.2.4 Procedure 75 

3.3 Pretreatment of the Extracted Phases 79 

References 81 

Bibliography 81 

Chapter 4 Mineralogical Characterisations by X-Ray Diffractometry 

4.1 Introduction 83 

4.1.1 X-Ray Diffraction and Mineralogy 83 

4.1.2 Principle 86 

4.1.3 XRD Instrumentation 87 

4.2 Qualitative Diffractometry 90 

4.2.1 Overview of Preparation of the Samples 90 

4.2.2 Preparation for Powder Diagrams 90 

4.2.3 Preparation for Oriented Diagrams 94 

4.2.4 Pretreatment of Clays 99 

4.2.5 Qualitative Diffractometry 113 

4.3 Quantitative Mineralogical Analysis 118 

4.3.1 Interest 118 

4.3.2 Quantitative Mineralogical Analysis by XRD 118 

4.3.3 Multi-Instrumental Quantitative Mineralogical Analysis 124 

References 1 26 

Bibliography 127 

General 127 

Preparation of Oriented Aggregates on Porous Ceramic Plate 128 

Saturation of Clays by Cations 129 

Saturation, Solvation, Intercalation Complex, Dissolution 129 

Preparation of I ron Oxides 1 30 

Quantitative XRD 130 

Chapter 5 Mineralogical Analysis by Infra-Red Spectrometry 

5.1 Introduction 133 

5.1.1 Principle 133 

5.1.2 IR Instrumentation 135 

5.2 IR Spectrometry in Mineralogy 138 

5.2.1 Equipment and Products 138 

5.2.2 Preparation of the Samples 139 

5.2.3 Brief Guide to Interpretation of the Spectra 146 

5.2.4 Quantitative Analysis 1 52 



Contents IX 

5.3 Other IR Techniques 156 

5.3.1 Near-infrared Spectrometry (NIRS) 156 

5.3.2 Coupling Thermal Measurements and FTIR Spectrometry of Volatile 
Products 158 

5.3.3 Infrared Microscopy 159 

5.3.4 Raman Scattering Spectroscopy 159 

References 161 

Chronobibliography 162 

Chapter 6 Mineralogical Separation by Selective Dissolution 

6.1 Introduction 167 

6.1.1 Crystallinity of Clay Minerals 167 

6.1.2 Instrumental and Chemical Methods 169 

6.1.3 Selective Dissolution Methods 172 

6.1.4 Reagents and Synthetic Standards 174 

6.2 Main Selective Dissolution Methods 180 

6.2.1 Acid Oxalate Method Under Darkness (AOD) 180 

6.2.2 Dithionite-Citrate-Bicarbonate Method (DCB) 187 

6.2.3 EDTA Method 192 

6.2.4 Pyrophosphate Method 196 

6.2.5 Extraction in Strongly Alkaline Mediums 201 

6.3 Other Methods, Improvements and Choices 206 

6.3.1 Differential Sequential Methods 206 

6.3.2 Selective Methods for Amorphous Products 210 

6.3.3 Brief Overview to the Use of the Differential Methods 214 

References 215 

Chapter 7 Thermal Analysis 

7.1 Introduction 221 

7.1.1 Definition 221 

7.1.2 Interest 223 

7.2 Classical Methods 226 

7.2.1 Thermogravimetric Analysis 226 

7.2.2 Differential Thermal Analysis and Differential Scanning Calorimetry 235 

7.3 Multi-component Apparatuses for Thermal Analysis 246 

7.3.1 Concepts 246 

7.3.2 Coupling Thermal Analysis and Evolved Gas Analysis 247 

References 249 

Chronobibliography 250 

Chapter 8 Microscopic Analysis 

8.1 Introduction 253 

8.2 Preparation of the Samples 254 

8.2.1 Interest 254 

8.2.2 Coating and Impregnation, Thin Sections 255 

8.2.3 Grids and Replicas for Transmission Electron Microscopy 261 

8.2.4 Mounting the Samples for Scanning Electron Microscopy 263 

8.2.5 Surface Treatment (Shadowing, Flash-carbon, Metallization) 265 



X Contents 

8.3 Microscope Studies 267 

8.3.1 Optical Microscopy 267 

8.3.2 Electron Microscopy, General Information 270 

8.3.3 Transmission Electron Microscopy, Micro-diffraction 271 

8.3.4 Scanning Electron Microscopy 279 

8.3.5 Ultimate Micro-analysis by X-Ray Spectrometry 282 

References 283 

Chronobibliography 284 

Part 2 - Organic analysis 



Chapter 9 Physical Fractionation of Organic Matter 

9.1 Principle and Limitations 289 

9.1.1 Forms of Organic Matter in Soil 289 

9.1.2 Principle 289 

9.1.3 Difficulties 291 

9.2 Methods 293 

9.2.1 Classification 293 

9.2.2 Extraction of Plant Roots 293 

9.2.3 Dispersion of the Particles 296 

9.2.4 Separation by Density 309 

9.2.5 Particle Size Fractionations 314 

9.2.6 Precision of the Fractionation Methods 320 

9.3 Conclusion and Outlook 321 

References 322 

Chapter 10 Organic and Total C, N (H, O, S) Analysis 

10.1 Introduction 327 

10.1.1 Soil Organic Matter 327 

10.1.2 Sampling, Preparation of the Samples, Analytical Significance 330 

10.2 Wet Methods 333 

10.2.1 Total Carbon: General Information 333 

10.2.2 Organic Carbon by Wet Oxidation at the Temperature 

of Reaction 335 

10.2.3 Organic Carbon by Wet Oxidation at Controlled Temperature 340 

10.2.4 Organic Carbon by Wet Oxidation and Spectrocolorimetry 342 

10.2.5 Total Nitrogen by Wet Method: Introduction 342 

10.2.6 Total Nitrogen by Kjeldahl Method and Titrimetry 344 

10.2.7 Kjeldahl N, Titration by Spectrocolorimetry 349 

10.2.8 Kjeldahl N, Titration by Selective Electrode 351 

10.2.9 Mechanization and Automation of the Kjeldahl Method 353 

10.2.10 Modified Procedures for N0 3 ~ N0 2 "and Fixed N 354 

10.3 Dry Methods 355 

10.3.1 Total Carbon by Simple Volatilization 355 

10.3.2 Simultaneous Instrumental Analysis by Dry Combustion: CHN(OS)356 

10.3.3 CHNOS by Thermal Analysis 362 



Contents XI 

10.3.4 C and N Non-Destructive Instrumental Analysis 363 

10.3.5 Simultaneous Analysis of the Different C and N Isotopes 364 

References 365 

Bibliography 367 

Chapter 1 1 Quantification of Humic Compounds 

11.1 Humus in Soils 371 

11.1.1 Definitions 371 

11.1.2 Role in the Soil and Environment 373 

11.1.3 Extractions 374 

11.2 Main Techniques 375 

11.2.1 Extraction 375 

11.2.2 Quantification of the Extracts 379 

1 1 .2.3 Precision and Correspondence of the Extraction Methods 383 

11.2.4 Purification of Humic Materials 389 

11.3 Further Alternatives and Complements Methods 392 

11.3.1 Alternative Method of Extraction 392 

11.3.2 Fractionation of the Humin Residue 392 

References 395 

Humic Materials 395 

Extraction, Titration, Purification and Fractionation of Humic Materials 396 

Chapter 12 Characterization of Humic Compounds 

12.1 Introduction 399 

12.1.1 Mechanisms of Formation 399 

12.1.2 Molecular Structure 400 

12.2. Classical Techniques 401 

12.2.1 Fractionation of Humic Compounds 401 

12.2.2 Titration of the Main Functional Groups 408 

12.2.3 UV-Visible Spectrometry 410 

12.2.4 Infra-Red Spectrography 413 

12.3 Complementary Techniques 415 

12.3.1 Improvements in Fractionation Technologies 415 

12.3.2 Titration of Functional Groups 418 

12.3.3 Characterization by Fragmentation 419 

12.3.4 Nuclear Magnetic Resonance (NMR) 424 

12.3.5 Fluorescence Spectroscopy 433 

12.3.6 Electron Spin Resonance (ESR) Spectroscopy 435 

12.3.7 Measurement of Molecular Weight and Molecular Size 437 

12.3.8 Microscopic Observations 440 

12.3.9 Other Techniques 441 

References 442 

Molecular Models 442 

Fractionation, Determination of Molecular Weights and Molecular Sizes .. 443 

Functional Group of Humic Compounds 445 

Spectrometric Characterizations 446 

UV-Visible, IR, Fluorescence, ESR Spectrometries 446 

Nuclear Magnetic Resonance 447 



XII Contents 

Methods of Characterization by Fragmentation 449 

Other Methods (Microscopy, X-ray, Electrochemistry, etc.) 451 

Chapter 13 Measurement of Non-Humic Molecules 

13.1 Introduction 453 

13.1.1 Non-Humic Molecules 453 

13.1.2 Soil Carbohydrates 453 

13.1.3 Soil Lipids 456 

1 3. 1 .4 Pesticides and Pollutants 457 

13.2 Classical Techniques 458 

13.2.1 Acid Hydrolysis of Polysaccharides 458 

13.2.2 Purification of Acid Hydrolysates 462 

13.2.3 Colorimetric Titration of Sugars 464 

13.2.4 Titration of Sugars by Gas Chromatography 467 

13.2.5 Quantification of Total Lipids 472 

13.2.6 Quantification of the Water-Soluble Organics 474 

13.3 Complementary Techniques 475 

13.3.1 Carbohydrates by Gas Chromatography 475 

13.3.2 Carbohydrates by Liquid Chromatography 475 

13.3.3 Fractionation and Study of the Soil Lipid Fraction 478 

13.3.4 Measurement of Pesticide Residues and Pollutants 483 

References 492 

Soil Carbohydrates 492 

Soil Lipids 494 

Aqueous Extract 495 

Pesticides and Pollutants 495 

Chapter 14 Organic Forms of Nitrogen, Mineralizable Nitrogen 
(and Carbon) 

14.1 Introduction 497 

14.1.1 The Nitrogen Cycle 497 

14.1.2 Types of Methods 499 

14.2 Classical Methods 500 

14.2.1 Forms of Organic Nitrogen Released by Acid Hydrolysis 500 

14.2.2 Organic Forms of Nitrogen: Simplified Method 509 

14.2.3 Urea Titration 511 

14.2.4 Potentially Available Nitrogen: Biological Methods 513 

14.2.5 Potentially Mineralizable Nitrogen: Chemical Methods 521 

14.2.6 Kinetics of Mineralization 526 

1 4.3 Complementary Methods 531 

14.3.1 Alternative Procedures for Acid Hydrolysis 531 

14.3.2 Determination of Amino Acids 532 

14.3.3 Determination of Amino Sugars 535 

14.3.4 Proteins and Glycoproteins (glomalin) 538 

14.3.5 Potentially Mineralizable Nitrogen by EUF 538 



Contents XIII 

References 540 

Organic Nitrogen Forms: General Articles 540 

Nitrogen Forms by Acid Hydrolysis and Distillation 541 

Improvement of Acid Hydrolysis 541 

Determination of Amino Acids 541 

Determination of Amino Sugars 542 

Glomalin 542 

Urea Titration 543 

Potentially Mineralizable Nitrogen: General Papers 543 

Potentially Mineralizable Nitrogen: Biological Methods 544 

Potentially Mineralizable Nitrogen: Chemical Methods 545 

Potentially Mineralizable Nitrogen by EUF 545 

Mineralization Kinetics 546 

Part 3 - Inorganic analysis - Exchangeable and Total Elements 

Chapter 15 pH Measurement 

15.1 Introduction 551 

15.1.1 SoilpH 551 

15.1.2 Difficulties 553 

15.1.3 Theoretical Aspects 554 

1 5.2 Classical Measurements 556 

15.2.1 Methods 556 

15.2.2 Colorimetric Method 557 

15.2.3 Electrometric Method 560 

15.2.4 Electrometric Checking and Calibration 564 

15.2.5 Measurement on Aqueous Soil Suspensions 565 

15.2.6 Determination of the pH-Kand pH-Ca 567 

15.2.7 Measurement on Saturated Pastes 567 

15.2.8 Measurement on the Saturation Extract 568 

15.2.9 Measurement of the pH-NaF 569 

15.3 In Situ Measurements 570 

15.3.1 Equipment 570 

15.3.2 Installation in the Field 570 

15.3.3 Measurement on Soil Monoliths 572 

References 574 

Bibliography bib 

Appendix 576 

Appendix 1: Table of Electrode Potentials 576 

Appendix 2: Constants of Dissociation of Certain Equilibriums 577 

Appendix 3: Buffer Solutions bll 

Appendix 4: Coloured Indicators 579 

Chapter 16 Redox Potential 

16.1 Definitions and Principle 581 

16.2 Equipment and Reagents 583 

16.2.1 Electrodes 583 

16.2.2 Salt Bridge for Connection 584 

16.2.3 System of Measurement 584 

16.2.4 Calibration Solutions 585 



XIV Contents 

16.3 Procedure 585 

16.3.1 Pretreatment of the Electrode 585 

16.3.2 Measurement on Soil Sample 586 

16.3.3 Measurement on Soil Monolith 586 

16.3.4 In Situ Measurements 587 

16.3.5 Measurement of Oxygen Diffusion Rate 588 

16.3.6 Colorimetric Test of Eh 589 

References 589 

Bibliography 590 

Chapter 17 Carbonates 

17.1 Introduction 593 

17.2 Measurement of Total Carbonates 595 

17.2.1 Introduction 595 

17.2.2 Volumetric Measurement by Calcimetry 596 

17.2.3 Acidimetry 599 

17.3 Titration of Active Carbonate 60 1 

17.3.1 Principle 601 

17.3.2 Implementation 601 

17.3.3 Index of Chlorosis Potential 603 

References 604 

Chapter 18 Soluble Salts 

18.1 Introduction 605 

18.2 Extraction 606 

18.2.1 Soil/solution Ratio 606 

18.2.2 Extraction of Saturated Paste 607 

18.2.3 Diluted Extracts 608 

18.2.4 In Situ Sampling of the Soil Water 609 

18.2.5 Extracts with Hot Water 610 

18.3 Measurement and Titration 610 

18.3.1 Electrical Conductivity of Extracts 610 

18.3.2 In Situ Conductivity 613 

18.3.3 Total Dissolved Solid Material 614 

18.3.4 Soluble Cations 615 

18.3.5 Extractable Carbonate and Bicarbonate (Alkalinity) 616 

18.3.6 Extractable Chloride 618 

18.3.7 Extractable Sulphate, Nitrate and Phosphate 620 

18.3.7 Extractable Boron 620 

18.3.8 Titration of Extractable Anions by Ionic Chromatography 622 

18.3.9 Expression of the Results 625 

References 626 

Chapter 19 Exchange Complex 

19.1 Introduction 629 

19.2 Origin of Charges 630 

19.2.1 Ionic Exchange 630 



Contents XV 

19.2.2 Exchange Complex 631 

19.2.3 Theory 633 

References 636 

Chronobibliography 637 

Chapter 20 Isoelectric and Zero Charge Points 

20.1 Introduction 645 

20.1.1 Charges of Colloids 645 

20.1.2 Definitions 647 

20.1.3 Conditions for the Measurement of Charge 649 

20.2 Main Methods 651 

20.2.1 Measurement of pHO (PZSE), Long Equilibrium Time 651 

20.2.2 Point of Zero Salt Effect (PZSE), Short Equilibrium Time 652 

References 655 

Chapter 21 Permanent and Variable Charges 

21.1 Introduction 657 

21.2 Main Methods 661 

21.2.1 Measurement of Variable Charges 661 

21.2.2 Determination of Permanent Charges 662 

References 664 

Bibliography 665 

Chapter 22 Exchangeable Cations 

22.1 Introduction 667 

22.1.1 Exchangeable Cations of Soil 667 

22.1.2 Extracting Reagents 668 

22.1.3 Equipment 669 

22.2 Ammonium Acetate Method at pH 7 671 

22.2.1 Principle 671 

22.2.2 Procedure 671 

22.3 Automated Continuous Extraction 674 

References 674 

Bibliography 676 

Chapter 23 Exchangeable Acidity 

23.1 Introduction 677 

23.1.1 Origin of Acidity 677 

23.1.2 Aims of the Analysis 678 

23.2 Method 680 

23.2.1 Principle 680 

23.2.2 Reagents 680 

23.2.3 Procedure 681 

23.3 Other Methods 683 

References 684 

Chronobibliography 685 



XVI Contents 

Chapter 24 Lime Requirement 

24.1 Introduction 687 

24.1.1 Correction of Soil Acidity 687 

24.1.2 Calculation of Correction 688 

24.2 SMP Buffer Method 690 

24.2.1 Principle 690 

24.2.2 Reagents 691 

24.2.3 Procedure 691 

24.2.4 Remarks 692 

References 693 

Chronobibliography 693 

Chapter 25 Exchange Selectivity, Cation Exchange Isotherm 

25.1 Introduction 697 

25.2 Determination of the Exchange Isotherm 702 

25.2.1 Principle 702 

25.2.2 Reagents 702 

25.2.3 Procedure 703 

25.2.4 Remarks 704 

References 705 

Chronobibliography 706 

Chapter 26 Cation Exchange Capacity 

26.1 Introduction 709 

26.1.1 Theoretical Aspects 709 

26.1 .2 Variables that Influence the Determination of CEC 71 1 

26.2 Determination of Effective CEC by Summation (ECEC) 718 

26.2.1 Principle 718 

26.2.2 Alternative Methods 718 

26.3 CEC Measurement at Soil phi in Not-Buffered Medium 719 

26.3.1 Principle 719 

26.3.2 Methods Using Not-Buffered Metallic Salts 719 

26.3.3 Procedure Using Not-Buffered Organo Metallic Cations 722 

26.3.4 Not-Buffered Methods Using Organic Cations 728 

26.4 CEC Measurement in Buffered Medium 730 

26.4.1 Buffered Methods — General Information 730 

26.4.2 Ammonium Acetate Method at pH 7.0 732 

26.4.3 Buffered Methods at pH 8.0-8.6 738 

26.4.4 Buffered Methods at Different pH 743 

References 745 

Bibliography 750 

CEC General Theory 750 

Barium Method at soil pH 751 

Buffered Method at pH 7.0 751 

Cobaltihexamine CEC 752 

Silver-Thiourea 753 

CEC with Organic Cations (Coloured Reagents) 753 

Buffered Methods at pH 8.0-8.6 753 

Barium Chloride-Triethanolamine at pH 8.1 753 



Contents XVII 

Chapter 27 Anion Exchange Capacity 

27.1 Theory 155 

27.2 Measurement 758 

27.2.1 Principle 758 

27.2.2 Method 760 

27.3 Simultaneous Measurement of AEC, EC, CEC and net CEC 760 

27.3.1 Aim 760 

27.3.2 Description 761 

References 763 

Chapter 28 Inorganic Forms of Nitrogen 

28.1 Introduction 767 

28.1.1 Ammonium, Nitrate and Nitrite 767 

28.1.3 Sampling Problems 768 

28.1.4 Analytical Problems 768 

28.2 Usual Methods 769 

28.2.1 Extraction of Exchangeable Forms 769 

28.2.2 Separation by Micro-Diffusion 770 

28.2.3 Colorimetric Titration of Ammonium 773 

28.2.4 Colorimetric Titration of Nitrites 115 

28.2.5 Colorimetric Titration of Nitrates 778 

28.2.6 Extracted Organic Nitrogen 779 

28.3 Other Methods 780 

28.3.1 Nitrate and Nitrite by Photometric UV Absorption 780 

28.3.2 Ammonium Titration Using a Selective Electrode 782 

28.3.3 Measurement of Nitrates with an Ion-Selective Electrode 785 

28.3.4 In situ Measurement 788 

28.3.5 Non-Exchangeable Ammonium 790 

References 791 

Bibliography 792 

Chapter 29 Phosphorus 

29.1 Introduction 793 

29.2 Total Soil Phosphorus 794 

29.2.1 Introduction 794 

29.2.2 Wet Mineralization for Total Analyses 795 

29.2.3 Dry Mineralization 798 

29.3 Fractionation of Different Forms of Phosphorus 799 

29.3.1 Introduction 799 

29.3.2 Sequential Methods 800 

29.3.3 Selective Extractions - Availability Indices 804 

29.3.4 Isotopic Dilution Methods 813 

29.3.5 Determination of Organic Phosphorus 814 

29.4 Retention of Phosphorus 818 

29.4.1 Introduction 818 

29.4.2 Determination of P Retention 819 



XVIII Contents 

29.5 Titration of P in the Extracts 821 

29.5.1 Introduction 821 

29.5.2 Titration of Ort/70-phosphoric P by Spectrocolorimetry 823 

29.5.3 P Titration by Atomic Spectrometry 828 

29.5.4 Titration of Different Forms of P by 31 P NMR 828 

29.5.5 Separation of P Compounds by Liquid Chromatography 829 

29.6 Direct Speciation of P in situ, or on Extracted Particles 830 

References 830 

Chronobibliography 833 

Chapter 30 Sulphur 

30.1 Introduction 835 

30.1.1 Sulphur Compounds 835 

30.1.2 Mineralogical Studies 838 

30.2 Total Sulphur and Sulphur Compounds 839 

30.2.1 Characteristics of Fluviomarine Soils 839 

30.2.2 Soil Sampling and Sample Preparation 840 

30.2.3 Testing for Soluble Sulphur Forms 841 

30.2.4 Titration of Total Sulphur 842 

30.2.5 Total S Solubilisation by Alkaline Oxidizing Fusion 843 

30.2.6 Total Solubilisation by Sodium Hypobromite in Alkaline Medium.... 844 

30.2.7 S titration with Methylen Blue Colorimetry 845 

30.2.8 Sulphate Titration by Colorimetry with Methyl Thymol Blue 850 

30.2.9 Total Sulphur by Automated Dry CHN(OS) Ultimate Analysis 853 

30.2.10 Titration of Total S0 4 2 "-S by Ionic Chromatography 855 

30.2.1 1 Total S Titration by Plasma Emission Spectrometry 857 

30.2.12 Titration by X-ray Fl uorescence 857 

30.2.13 Titration by Atomic Absorption Spectrometry 857 

30.2.14 Analytical Fractionation of Sulphur Compounds 858 

30.2.15 Titration of Organic S bound to C 859 

30.2.16 Titration of Organic S not bound to C 861 

30.2.17 Extraction and Titration of Soluble Sulphides 863 

30.2.18 Titration of Sulphur in Pyrites 865 

30.2.19 Titration of Elementary Sulphur 867 

30.2.20 Titration of Water Soluble Sulphates 869 

30.2.21 Titration of Na 3 -EDTA Extractable Sulphates 871 

30.2.22 Titration of Jarosite 873 

30.2.23 Sequential Analysis of S Forms 876 

30.3 Sulphur of Gypseous Soils 878 

30.3.1 Gypseous Soils 878 

30.3.2 Preliminary Tests 879 

30.3.3 Extraction and Titration from Multiple Extracts 881 

30.3.4 Gypsum Determination by Acetone Precipitation 882 

30.4 Sulphur and Gypsum Requirement of Soil 883 

30.4.1 Introduction 883 

30.4.2 Plant Sulphur Requirement 884 

30.4.3 Gypsum Requirement 886 

References 888 

Chronobibliography 890 



Contents XIX 

Chapter 31 Analysis of Extractable and Total Elements 

31.1 Elements of Soils 895 

31.1.1 Major Elements 895 

31.1.2 Trace Elements and Pollutants 897 

31.1.3 Biogenic and Toxic Elements 899 

31.1.4 Analysis of Total Elements 900 

31.1.5 Extractable Elements 901 

31.2 Methods using Solubilization 901 

31.2.1 Total Solubilization Methods 901 

31.2.2 Mean Reagents for Complete Dissolutions 903 

31.2.3 Acid Attack in Open Vessel 906 

31.2.4 Acid Attack in Closed Vessel 911 

31.2.5 Microwave Mineralization 913 

31.2.6 Alkaline Fusion 915 

31 .2.7 Selective Extractions 920 

31.2.8 Measurement Methods 925 

31.2.9 Spectrocolorimetric Analysis 927 

31.2.10 Analysis by Flame Atomic Emission Spectrometry 931 

31 .2.1 1 Analysis by Flame Atomic Absorption Spectrometry 932 

31.2.12 Analysis of Trace Elements by Hydride and Cold Vapour AAS 937 

31.2.13 Analysis of Trace Elements by Electrothermal AAS 940 

31.2.14 Analysis by Inductively Coupled Plasma-AES 941 

31 .2.15 Analysis by Inductively Coupled Plasma-Mass Spectrometry 946 

31.3 Analysis on Solid Medium 952 

31.3.1 Method 952 

31.3.2 X-ray Fluorescence Analysis 954 

31.3.3 Neutron Activation Analysis 962 

References 969 

Index 975 

periodic table of the elements 993 



Parti 



Mineralogical 
Analysis 



Water Content and Loss on Ignition 



1.1 Introduction 

Schematically, a soil is made up of a solid, mineral and organic phase, a 
liquid phase and a gas phase. The physical and chemical characteristics of 
the solid phase result in both marked variability of water contents and a 
varying degree of resistance to the elimination of moisture. 

For all soil analytical studies, the analyst must know the exact quantity 
of the solid phase in order to transcribe his results in a stable and 
reproducible form. The liquid phase must be separate, and this operation 
must not modify the solid matrix significantly (structural water is related 
to the crystal lattice). 

Many definitions exist for the terms "moisture" and "dry soil". The 
water that is eliminated by moderate heating, or extracted using solvents, 
represents only one part of total moisture, known as hygroscopic water, 
which is composed of (1) the water of adsorption retained on the surface 
of solids by physical absorption (forces of van der Waals), or by 
chemisorption, (2) the water of capillarity and swelling and (3) the 
hygrometrical water of the gas fraction of the soil (ratio of the effective 
pressure of the water vapour to maximum pressure). The limits between 
these different types of water are not strict. 

"Air-dried" soil, which is used as the reference for soil preparation in 
the laboratory, contains varying amounts of water which depend in 
particular on the nature of secondary minerals, but also on external forces 
(temperature, the relative humidity of the air). Some andisols or histosols 
that are air dried for a period of 6 months can still contain 60% of water 
in comparison with soils dried at 105°C, and this can lead to unacceptable 
errors if the analytical results are not compared with a more realistic 



Mineralogical Analysis 



reference for moisture. 1 Saline soils can also cause problems because of 
the presence of hygroscopic salts. 

It is possible to determine remarkable water contents involving fields of 
force of retention that are sufficiently reproducible and representative 
(Table 1.1). These values can be represented in the form of capillary 
potential (pF), the decimal logarithm of the pressure in millibars needed 
to bring a sample to a given water content (Table 1.1). It should be noted 
that because of the forces of van der Waals, there can be differences in 
state, but not in form, between water likely to evaporate at 20°C and 
water that does not freeze at -78°C. The analyst defines remarkable 
points for example: 

- The water holding capacity, water content where the pressure 
component of the total potential becomes more significant than the 
gravitating component; this depends on the texture and the nature of the 
mineral and approaches field capacity which, after suitable drainage, 
corresponds to a null gravitating flow. 

- The capillary frangible point, a state of moisture where the continuous 
water film becomes monomolecular and breaks. 

- The points of temporary and permanent wilting where the pellicular 
water retained by the bonding strength balances with osmotic pressure; 
in this case, except for some halophilous plants, the majority of plants 
can no longer absorb the water that may still be present in the soil. 

- The hygroscopic water which cannot be easily eliminated in the natural 
environment as this requires considerable energy, hygroscopic water 
evaporates at temperatures above 100°C and does not freeze at -78°C. 

- The water of constitution and hydration of the mineral molecules can 
only be eliminated at very high pressures or at high temperatures, with 
irreversible modification or destruction of the crystal lattice. 

These types of water are estimated using different types of 
measurements to study the water dynamics and the mechanisms related to 
the mechanical properties of soils in agronomy and agricultural 
engineering, for example: 

- usable reserves (UR), easily usable reserves (EUR), or reserves that are 
easily available in soil-water-plant relations. 

-thresholds of plasticity, adhesiveness, liquidity (limits of Atterberg, 
etc.). 



1 It should be noted that for these types of soil, errors are still amplified by the 
ponderal expression (because of an apparent density that is able to reach 0.3) 
this is likely to make the analytical results unsuitable for agronomic studies. 



Water Content and Loss on Ignition 



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£ R 

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Water of Hygroscopic water Water of swelling Capillary water 

constitution" evaporation 100°C - evaporation > 100°C 
- does not freeze at -78°C. freezes at -1 to -78°C 

* * ■— * 



Water of gravity 

- evaporation 20°C Excess 

- congelation - 1°C water 

t H 



g PQ 

Ph 



Slow flow 
USEFUL WATER 



Rapid flow 



AVAILABLE WATER (B) 



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Mineralogical Analysis 



This brief summary gives an indication of the complexity of the 
concept of soil moisture and the difficulty for the analyst to find a 
scientifically defined basis for dry soil where the balance of the solid, 
liquid and gas phases is constant. 



1.2 Water Content at 105°C (H 2 0) 



1.2.1 Principle 

By convention, the term "moisture" is considered to be unequivocal. 
Measurement is carried out by gravimetry after drying at a maximum 
temperature of 105°C. This increase in temperature maintained for a 
controlled period of time, is sufficiently high to eliminate "free" forms of 
water and sufficiently low not to cause a significant loss of organic matter 
and unstable salts by volatilization. Repeatability and reproducibility are 
satisfactory in the majority of soils if procedures are rigorously respected. 



1.2.2 Materials 

- 50 x 30 mm borosilicate glass low form weighing bottle with ground 
flat top cap. 

- Vacuum type 200 mm desiccator made of borosilicate glass with 
removable porcelain floor, filled with anhydrous magnesium 
perchlorate [Mg(C104)2]. 

- Thermostatically controlled drying oven with constant speed blower for 
air circulation and exhausting through a vent in the top of oven - 
temperature uniformity ± 0.5-1 °C. 

- Analytical balance: precision 0.1 mg, range 100 g. 

1.2.3 Sample 

It is essential to measure water content on the same batch of samples 
prepared at the same time (fine earth with 2 mm particles or ground soil) 
for subsequent analyses. It should be noted that the moisture content of 
the prepared soil may change during storage (fluctuations in air moisture 
and temperature, oxidation of organic matter, loss or fixing of volatile 
substances, etc.). 



Water Content and Loss on Ignition 



This method can be considered "destructive" for certain types of soils 
and analyses, as the physical and chemical properties can be transformed. 
Samples dried at 105°C should generally not be used for other 
measurements. 



1.2.4 Procedure 

-Dry tared weighing bottles for 2 h at 105°C, let them cool in the 
desiccator and weigh the tare with the lid placed underneath: m 

- Place about 5 g of air-dried soil (fine earth sieved through a 2 mm 
mesh) in the tare box and note the new weight: m\ 

- Place the weighing bottles with their flat caps placed underneath in a 
ventilated drying oven for 4 h at 105°C (the air exit must be open and 
the drying oven should not be overloaded) 

- Cool in the desiccator and weigh (all the lids of the series contained in 
the desiccator should be closed to avoid moisture input): m 2 

- Again place the opened weighing bottles in the drying oven for 1 h at 
105°C and weigh under the same conditions; the weight should be 
constant; if not, continue drying the weighing bottles until their weight 
is constant 



% water content at 105°C = 100 



m x —m 2 

m x -m 



1.2.5 Remarks 

The results can also be expressed in pedological terms of water holding 

capacity (HC) by the soil: HC = 100 x ™ l ~ ^ 2 . 

m 2 -m Q 

The point of measurement at 105°C with constant mass is empirical 
(Fig. 1.1). A temperature of 130°C makes it possible to release almost all 
"interstitial water", but this occurs to the detriment of the stability of 
organic matter. The speed of drying should be a function of the 
temperature, the surface of diffusion, the division of the solid, ventilation, 
pressure (vacuum), etc. 

Respecting the procedure is thus essential: 

- For andisols and histosols, the initial weighing should be systematically 
carried out after 6 h. 

- For saline soils with large quantities of dissolved salts, the sample can 
be dried directly, soluble salts then being integrated into the "dry soil" 
or eliminated beforehand by treatment with water. 



Mineralogical Analysis 



Fig. 1.1 -Theoretical 

diagrammatic curve 
showing water moved at 
a given temperature as a 
function of time (180°C = 
end of H 2 losses in 
allophanes) 



CO 
CO 

O 

CO 
CO 
03 




180°C 

130°C 
105°C 



1.3 Loss on Ignition at 1,000°C (H 2 + ) 

1.3.1 Introduction 

As we have just seen, the reference temperature (105°C) selected for the 
determination of the moisture content of a "dry soil" represents only a 
totally hypothetical state of the water that is normally referred to as H20~ 
When a sample undergoes controlled heating and the uninterrupted 
ponderal variations are measured, curves of "dehydration" are obtained 
whose inflections characterize losses in mass at certain critical 
temperatures (TGA). 1 If one observes the temperature curve compared to 
a thermically inert substance (Fig. 1.2), it is possible to determine 
changes in energy between the sample studied and the reference 
substance, this results in a change in the temperature which can be 
measured (DTA-DSC). 2 

- If the temperature decreases compared to the reference, an endothermic 
peak appears that characterizes loss of H2O (dehydration), of OFT 
(dehydroxylation), sublimation, or evaporation, or decomposition of 
certain substances, etc. 

- If the temperature increases compared to the reference, an exothermic 
peak appears that characterizes transformations of crystalline structures, 
oxidations (Fe 2+ — > Fe 3+ ), etc. 



2 TGA thermogravimetric analysis; DTA differential thermal analysis; DSA 
differential scanning calorimetry (cf. Chap. 7). 



Water Content and Loss on Ignition 



< 



to 

w 



Fig. 1.2 - Schematized 

example of thermal 
analysis curves 
TGA (solid line) and 
DTA (dashed line) 




TGA 



Kaollnite 



DTA 

v 
Adsorbed 

wate 



Free 
water 



Mon:morill')nite 



Linkec 
water 



A 



V I 



Stable residue 



Linked 
water 



__j 



i 



Sable 
residue 



o 
5! 



200 400 600 



800 1000 
T(°C) 



The simultaneous analysis of the gases or vapours that are emitted and 
X-ray diffraction (cf. Chap. 4) of the modifications in structure make it 
possible to validate the inflections of the curves or the different endo- and 
exothermic peaks. 

As can be seen in the highly simplified Table 1.2, the most commonly 
observed clays are completely dehydroxyled at 1,000°C, oxides at 400°C 
or 500°C, carbonates, halogens, sulphates, sulphides are broken down or 
dehydrated between 300°C and 1,000°C, and free or bound organic 
matter between 300°C and 500°C. The temperature of 1,000°C can thus 
be retained as a stable reference temperature for loss on ignition, the 
thermal spectra then being practically flat up to the peaks of fusion which 
generally only appear at temperatures higher than 1,500°C or even 
2,500°C. 



1.3.2 Principle 

The sample should be gradually heated in oxidizing medium to 1,000°C 
and maintained at this temperature for 4 h. 



10 



Mineralogical Analysis 



Table 1.2 Dehydration and dehydroxylation of some clays, oxides and 
salts as a function of temperature in °C 



type 


name 


dehydration a 


dehydroxylation b 


clays 1:1 


Kaolinite-halloysite 


350 


1,000 


clays 2:1 


smectites - 
montmorillonite 


370 


1,000 


clays 2:1 


Illite - micas 


350-370 


1,000 


clays 2:1 


vermiculite 


700 


1,000 


clays 2:1:1 


chlorite 


600 


800 


fibrous clays 


Sepiolite- 

palygorskite 

allophane 


300 
200 


800-900 
900-1,000 


iron oxides 


Hematite a Fe203 


(flat 
spectrum) 


1,000 




goethite a FeO-OH 


100 


370 




magnetite Fe203 


375 


650 


Al oxides 


gibbsite y-Al(OH)3 


100 


350 


Ca carbonate 


Calcite-aragonite 
CaC03 


— 


950-1,000 


Mg carbonate 


magnesite MgC03 


- 


710 


Ca-Mg 
carbonate 


dolomie 
CaMg(C03)2 


— 


800-940 


halogenous 
compounds 


sodium chloride 
NaCl 


— 


800 (fusion) 


sulphate 


gypsum CaS04, 
2H 2 


— 


300 


sulphide 


pyrite FeS2 


- 


615 


organic 
compounds 


free or linked organic 
matter 


— 


300-500 



a Dehydration: loss of water adsorbed on outer or inner surfaces, with 
or without reversible change in the lattice depending on the types of 
clay, water organized in monomolecularfilm on surface oxygen 
atoms or around exchangeable cations. 

b dehydroxylation (+ decarbonatation and desulphurization reactions), 
loss of water linked to lattice (OH"), irreversible reaction or 
destruction of the structure, water present in the cavities, O forming 
the base of the tetrahedrons. 



Water Content and Loss on Ignition 1 1 

Loss on ignition is determined by gravimetry. It includes combined 
water linked to the crystal lattice plus a little residual non-structural 
adsorbed water, organic matter, possibly volatile soluble salts (F~, S 2 ") 
and carbonates (C0 3 2 ~, C0 2 ). The use of an oxidizing atmosphere is 
essential to ensure combustion of the organic matter and in particular 
oxidation of reduced forms of iron, this being accompanied by an 
increase in mass of the soils with minerals rich in Fe 2+ . A complete 
analysis generally includes successive measurements of H 2 0~ and H 2 + 
on the same sample. 

1.3.3 Equipment 

- Platinum or Inconel (Ni-Cr-Fe) crucible with cover, diameter 46 mm. 

- Analytical balances (id. H 2 0~) 

- Desiccator (id. H 2 0") 

-Muffle electric furnace (range 100-1,100°C) with proportional 
electronic regulation allowing modulation of the impulses with 
oscillation of about 1°C around the point of instruction; built-in 
ventilation system for evacuation of smoke and vapour 

- Thermal protective gloves 

- 300 mm crucible tong 

1.3.4 Procedure 

- Tare a crucible, heat it to 1 ,000°C and cool it in the desiccator with its 
lid on: m 

- Introduce 2-3 g of air-dried soil crushed to 0.1 mm: m l 

- Dry in the drying oven at 105°C for 4 h 

- Cool in the desiccator and weigh: m 2 

- Adjust the lid of the crucible so it covers approximately 2/3 of the 
crucible and put it in the electric furnace 

- Programme a heating gradient of approximately 6°C per minute with a 
20-min stage at 300°C, then a fast rise at full power up to 1,000°C with 
a 4-h graduation step (the door of the furnace should only be closed 
after complete combustion of the organic matter) 

- Cool the crucible in the desiccator and weigh: m 3 



1.3.5 Calculations 

mi -mo = weight of air-dried soil 
mi -m 2 = moisture at 105°C 



12 Mineralogical Analysis 

m 2 - m = weight of soil dried at 105°C 

m 2 -m 3 = loss on ignition 



H 2 CT% = 100 x 



H + % = 100: 



1.3.6 Remarks 



Mi 


~ m 2 


m l 


-m 


m 2 


-m 3 



m>-> -ma 



related to air-dried soil 
related to soil dried at 105°C 



Knowing the moisture of the air-dried soil, it is possible to calculate the 
weight of air-dried soil required to work with a standard weight soil dried 
at 105°C, thus simplifying calculations during analyses of the samples. 

To obtain the equivalent of 1 g of soil dried at 105°C, it is necessary to 
weigh: 

100 

with wc = % water content of air dried soil. 

100 -wc 

Platinum crucibles are very expensive and are somewhat volatile at 
1,000°C, which means they have to be tared before each operation, 
particularly when operating in reducing conditions. 

Combustion of organic matter with insufficient oxygen can lead to the 
formation of carbide of Pt, sulphides combine with Pt, chlorine attacks Pt, 
etc. 



Bibliography 

Campbell GS, Anderson RY (1998) Evaluation of simple transmission line 

oscillators for soil moisture measurement. Comput. and Electron. Agric, 

20,31-44 
Chin Huat Lim, Jackson ML (1982) Dissolution for total elemental analysis. In 

Methods of Soil Analysis, Part 2, Page A.L., Miller R.H., Kenny D.R. ed. 

Am. Soc. Agronomy, pp. 1-11 
Dixon JB (1977) Minerals in soil environments. Soil Sci. Soc. Am. 
Dubois J, Paindavoine JM (1982) Humidite dans les solides, liquides et gaz. 

Techniques de ringenieur.,(F 3760) 
Gardner WH (1986) Water content. In Methods of Soil Analysis, Part 1, Klute 

ed. Am. Soc. Agronomy, Soil Sci. Soc. Am., pp. 493-544 
Henin S (1977) Cours de physique du sol: Veau et le sol tome II., Editest, Paris: 

1-164 
Lane PNJ, Mackenzie DH, Nadler AD (2002) Note of clarification about: Field 

and laboratory calibration and test of TDR and capacitance techniques for 

indirect measurement of soil water content. Aust. J. Soil Res., 40, 

555-1386 



Water Content and Loss on Ignition 13 

Lane PNJ, Mackenzie DH (2001) Field and laboratory calibration and test of 

TDR and capacitance techniques for indirect measurement of soil. Aust. 

J. Soil Res., 39, 1371-1386 
NF ISO 11465 (X3 1-102) (1994) Determination de la teneur ponderale en 

matiere seche et en eau. In Qualite des sols, AFNOR, 1996, 517-524 
Rankin LK, Smaj stria AG (1997) Evaluation of the carbide method for soil 

moisture measurement in sandy soils. Soil and Crop Science Society of 

Florida, 56, pp. 136-139 
Skierucha W (2000) Accuracy of soil moisture measurement by TDR technique. 

Int. Agrophys., 14,417-426 
Slaughter DC, Pelletier MG, Upadhyaya SK (2001) Sensing soil moisture using 

NIR spectroscopy. Appl. Eng. Agric, 17, 241-247 
Walker JP, Houser PR (2002) Evaluation of the Ohm Mapper instrument for soil 

moisture measurement. Soil Sci. Soc. Am. J., 66, 728-734 
X3 1-505 (1992) Methode de determination du volume, apparent, et du contenu 

en eau des mottes. In Qualite des sols, AFNOR, 1996, 373-384 
Yu C, Warrick AW, Conklin MH (1999) Derived functions of time domain 

reflectometry for soil moisture measurement. Water Resour. Res., 35, 

1789-1796 



Particle Size Analysis 



2.1 Introduction 



2.1.1 Particle Size in Soil Science 

Determination of grain-size distribution of a sample of soil is an important 
analysis for various topics in pedology, agronomy, sedimentology, and other 
fields such as road geotechnics. 

Soil texture has an extremely significant influence on the physical and 
mechanical behaviours of the soil, and on all the properties related to 
water content and the movement of water, (compactness, plasticity, thrust 
force, slaking, holding capacity, moisture at different potentials, per- 
meability, capillary movements, etc.). 

Particle size analysis of a sample of soil, sometimes called "mechanical 
analysis", is a concept that has been the subject of much discussion 
(Henin 1976). Soil is an organized medium including an assemblage of 
mineral and organic particles belonging to a continuous dimensional 
series. The first difficulty is to express the proportion of these different 
particles according to a standard classification, which is consequently 
somewhat artificial. 

One classification scale was proposed by Atterberg (1912). Today this 
scale is recognized at different national and international levels and 
includes two main fractions: fine earth (clay, silts and sands with a grain 
diameter <2 mm) and coarse elements (gravels, stones with a grain 
diameter >2 mm). The particle size series (Fig. 2.1) for fine earth is 
generally expressed after analysis in three size fractions (clay fraction 
less than 0.002 mm, silt fraction from 0.002 to 0.02 mm, and sand 
fraction from 0.02 to 2 mm). In some countries, or for the purpose of a 
particular type of pedological interpretation, a more detailed scale of 
classes is sometimes used, for example five fractions: fine clays, silts, 
coarse silts or very fine sands, fine sands, and coarse sands (Fig. 2.1). 



16 



Mineralogical Analysis 



1 urn 


1 2 M m 1020 SO 


1 00 200 wm 500 Fine earth Coarse etaments 


NC 


% 


II 


10 002 (003i 0.1 02 05 1mm 2mm 20mm75 


mm 250 mm 


0,0002 


005 0.05 010 0.25 0.50 100 


00 




FC 


Coarse 
clays 


FSI 


Medium 

SillS 


to 


{A 
1 


Fine 
sands 


MS 


Coarse 
sands 


Very coarse 
sands 


Gravete 


Stones 


10 


U 

w 

o 


002 0.02 0. OS 0.10 020 SO 1 00 


00 




Clays 


r ihq 

SUES 


03 


FS 
<1> 


Fine 

sands 

(2) 


cs 
(1> 


Coarse 
sands 

m 


Coarse 

sands 

0) 




e 


8 

E 


0.002 0.050,10 0,25 0.50 ? 


00 




Clays 


Silts 


CO 


Fine 
sands 


MS 


Coarse 
sands 


Very 
coarse 
sands 


Gravels 
Fine | Coarse 


*~ 


7 










Clays 


Fine 
silts 





Fine 

sands 


Coarse 
sands 


| 




5 


1 
Si 


0002 0.02 20 2 


00 




Days 
(I) 


Silts 


Fine sands {111] 
S^S,: FS, 


Coarse sands (IV) 
CS, ; cs ? | cs, 


Graves 


ISSS 
Attcrbero 


A 


1 








Fines 
[silts and diys) 


Fine 
sands 


Medium sands 


s 


u-, CG 


Stones 


3 


3 
& 



200 


35 


10 1/2 


3 « Inch or standard 


75 jim 


500 |im 


2 mm 


ASTM 



Fig. 2.1. Ranges of particle size used for soils (NC number of classes; FSi fine 
silts, CSi coarse silts; FS, VFS, CS fine, very fine and coarse sands, 
respectively; FC fine clays; FG, CG fine gravels and coarse gravels), 
from top to bottom: (CSSC) Canadian Soil Survey Committee (1978): 10 
particle size ranges < 2 mm; France (before 1987): 8 ranges; USDA 
United States Department of Agriculture (1975): 7 ranges; AFNOR 
Association Frangaise de Normalisation (1987): 5 ranges; ISSS 
= International Soil Science Society (1966): 4 ranges; ASTM = American 
Society for Testing Materials (1985): 3 ranges 



However, it should be noted that the terminology used does not provide 
much information about the real nature of the classes; thus clay defined as 
having a diameter equal to or less than 0.002 mm does not contain only 
clay corresponding to this mineralogical definition but can also contain 
sesquioxides, very fine silts, organic matter, carbonates, or compounds 
without colloidal properties. In the same way, sands, which generally result 
from fragmentation of the parent rock, can also include pseudo-sands, small 
ferruginous concretions, small limestone or cemented nodules that are 
resistant to dispersion treatments. The presence of these pseudo-sands can 
render the conclusions of particle size analysis illusory. 

Another difficulty appears with the fractionation of elementary 
particles by dissociating them from their original assembly. Here too 
analytical standards exist, but it should be recognized that in certain cases 
the rupture of all the forces of cohesion is not complete (the case in 
hardened cemented soils), or on the contrary the forces are too energetic. 

Lastly, particle size analysis accounts for the size but not for the shape 
of the particles, or their nature. If necessary, these are the subject of 



Particle Size Analysis 17 

specific morphoscopic and mineralogical analyses. The result of particle 
size analysis is expressed in classes of which the relative proportions can 
be summed up in the form of a triangular diagram enabling the texture of 
a sample, a horizon, or a soil to be defined. Depending on the school, 
there are several different types of triangles that represent textures: 
GEPPA (Groupe d'Etude des Problemes de Pedologie Appliquee, AFES, 
Grignon, France) includes 17 textural classes; the USDA's (United States 
Department of Agriculture) includes 12 classes (Gras 1988); others are 
simplified to a greater or lesser extent depending on the pedological or 
agronomic purpose of the study. Starting from these results, different 
interpretations are usually made in terms of pedogenesis (comparison of 
the vertical sand percents to check the homogeneity of a given material in 
a given soil profile, calculation of different indices of leaching, clay 
transport, etc.); others are more practical (definition of the relation of 
texture to hydric characteristics for the initial calculation of the amounts 
and frequencies of irrigation, or for the choice of machinery for 
cultivation. 



2.1.2 Principle 

Particle size analysis is a laboratory process, which initially causes 
dissociation of the material into elementary particles; this implies the 
destruction of the aggregates by eliminating the action of cements. But 
this action should not be too violent to avoid the creation of particles that 
would not naturally exist; the procedure of dispersion must thus be 
sufficiently effective to break down the aggregates into individual 
components, but not strong enough to create neo-particles. 

Measurements (Table 2.1, Fig. 2.2) then will link the size of the 
particles to physical characteristics of the suspension of soil after 
dispersion (cf. Sect. 2.1.3). These measurements may be distorted by the 
presence of some compounds in the soil: organic matter, soluble salts, 
sesquioxides, carbonates, or gypsum. The latter compound can be 
particularly awkward because it can result in two opposing actions 
(Vieillefon 1979): flocculation due to soluble calcium ions (relative 
reduction in clay content), and low density of gypsum compared to other 
minerals (increase in clay content). Particle size analysis thus generally 
starts with a pre-treatment of the sample that varies with the type of soil; 
the characteristics of different soils are given in Table 2.3. 



18 Mineralogical Analysis 

1 ,0.2 1_ 2 5 jam 10 ,50 ^m 100 200 500 [am 1 mm 2, mm 20 mm 



Divers Sedimentation Sieving ult ra-sons Sieving : Manual shifting 



1- 150 | um Elutriation 

1 - 1 50 urn Sedimentation bala nces 

0.001 ~5 fim Malvern Zetasizer 

3nm-3um Malvern 4600 



0l01 0.5^560Mm - Malvern Autosizer Mg|vem ^ £ 

0.1 -2000 urn 8 P 1 , 30 °°J Malvern MastersizerX (4 ranges) 

— 0-1 -600 [am Malvern Mastersizer x (3 ranges) 



Ituwpn Malvern 2600 short 



3 nm - 3 [im 

Malvern HiC Coulter N4 



Malvern 2600 long 



0.04 - 3 ^m 



Coulter NaNoSizer 



61500 ,,m ^ouhbi .Nd.Nuo.^1 Coulter TAN 



3-570[gm Coulter LS130 

0-004-2000 iim CILAS-ALCATEL (1996) 

0.7- 400 urn x 

0.1- 500 [im 

10nm-5^m NiconpTClOO 

1- 200 u rn - 50 M m 



Analysette 20 Fristsh 

Micromeritics Sedigraph 5100 
0.04 -1 000 jum~; Micromeritics Sedigraph 5000 E MorJba LAgoo 



0.1-300 n m 



0.1-150 Mm 



).Q2-5Qnm -100 u rn 

Moriba Capa 500 



Shimadzu SA Cl 2 10 -SACI 2 20 

0,01-30 ma Joyce-Loebl Mark III 

0.01-60 [am 



X-ray UVVISIBLE |f Joyce-Loebl DC-3 



J _L 



Infra-Red 



0.01 fim 0.4 u.m 1 |um 



Fig. 2.2. Particle size ranges of some automated particle-measurement instruments 



2.1.3 Law of Sedimentation 

After possible pretreatment (cf. Sect. 2.2.1), the sample is suspended in 
aqueous medium in the presence of a dispersant (cf. Sect. 2.2.2). During 
sedimentation, the particles are then subjected to two essential forces: a 
force of gravity that attracts them to the bottom, and a force of viscous 
resistance of the medium in the opposite direction to their displacement. 
By comparing the particles to spheres of radius r, the force of gravity F g 
(dynes) is expressed by: 

F g = - Kr3 {ps-Pf)g 

r = equivalent radius of the spherical particle in cm; 



-2. 



g = gravity constant, 981 cm s 

p s = density of the particles in g cm" 3 (between 2.4 and 2.8 for soils); 



-3. 



pf = density of the liquid of dispersion in g cm 

The force of resistance of the medium F r (dynes) is expressed by: 



Particle Size Analysis 19 

F Y = 6 71 r r\ V, 

V= falling speed in cm s" 1 ; 

7] = viscosity of the medium in Poises (g cm" 1 s" 1 ), at temperature 
0°C (Table 2.2). 

When the particles reach equilibrium, the forces F T and F T are equal, 
from which their drop speed can be estimated according to the law 
originally established by Stokes (1851): 

9 ri 

For calculations, the average density of the solid particles in 
dispersions of soils is often selected with p s = 2.65 or 2.60 g cm" 3 . 
Empirical relationships have been established for the calculation of p F 
and 7] in aqueous solutions of hexametaphosphate generally used for 
particle-size distribution of soils (Gee and Bauder 1986): 

p t = p (1 + 0.630 C HM p), (2.2) 

7] = % (1 + 4.25 C HM p), (2.2') 

p = density of water (g cm" 3 at the working temperature (Table 2.2); 
r/ = viscosity of water (poise) at the working temperature (Table 2.2); 
Chmp = hexametaphosphate concentration in g cm" 3 
The constant of Stokes for the medium can thus be defined by: 

C = 2(p s -p f )g/9 7j. 

Equation (2.1) shows that the falling speed is proportional to the 
square of the particle radius and remains constant throughout 
sedimentation if certain conditions are strictly respected (cf. Sect. 2.1.4). 
The speed can also be defined by V = h/t where T is the time (s) spent by 
the particle of radius r(cm) to fall a height //(cm). Either the depth of its 
sedimentation over a given period, or the time needed for sedimentation 
to a given depth is determined by: 

9 h 77 i o 

t = \ = h C" 1 r~\ (2.3) 

2k " Pf)gr 



20 



Mineralogical Analysis 



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Particle Size Analysis 



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Particle Size Analysis 



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24 Mineralogical Analysis 

Particle size analysis by sedimentation consists in determining the 
content of particles below or equal to a given threshold. Known volumes 
of solution (pipette method) are generally used for the depth and time of 
sedimentation chosen as the threshold for a cut point. After drying the 
pipette sample, weighing and correcting the volume, the content of 
particles that are smaller than the selected threshold can be determined. In 
the example in Table 2.2, a pipette sample at a temperature of 20°C, a 
depth of 10 cm and 8 h 08 min of sedimentation will give the content of 
the clay fraction (diameter of particle < 2 |Lim). 

In the densimetry method, the relation between the size of the particles 
(radius r) and the time of sedimentation t can be expressed by: 

r = St~ l, \ (2.4) 

where S is the parameter of sedimentation. Taking into account (2.3), it 
can be expressed by: 

S=C~ m H r m , (2.4') 

where H r is the depth of balance of the densimeter (hydrometer) which 
represents the effective measurement depth of the particles of radius r. 



2.1.4 Conditions for Application of Stokes Law 

The formula of Stokes is theoretically only valid for particles with a 
diameter of less than 0.1 mm, but according to Meriaux (1954), it can be 
used up to 0.2 mm or even 0.05 mm. Above this value, it is advisable to 
apply the formula of Oseen; however, particles more than 0.1 mm in 
diameter can be more precisely sorted by sieving. 

For particle size analysis, sedimentation cylinders are used whose 
walls slow down the falling speed of the particles by friction. Thus, for 
0.05 mm quartz spheres, the falling speed at a distance of 0.1 mm from 
the walls is reduced by 12%, and disturbance becomes negligible at 1 mm 
(0.28%). In practice, it may be advantageous to use sedimentation tubes 
(cylinders) with a rather large diameter, at least 5 cm. 

In addition, the constant of Stokes is established for minerals with an 
average density of 2.60 or 2.65, whereas soil materials can contain illite 
with a density of 2.1 - 2.7, montmorillonite with a density of 1.7 - 2.6 
and so on. But the main difficulty is the fact that the particles are neither 
spherical, nor smooth, which obliges the analyst to introduce the concept 
of equivalent radius. 



Particle Size Analysis 



25 



Table 2.2. Densities p and viscosities r/ of water and 5% hexametaphosphate 
solutions related to temperature 6 (°C); corresponding to Stoke C 
constants and falling time t at 10 cm depth for clay particles > 2 jim 
(p s = 2.60 g cm" 3 ) in hexametaphosphate solution 

















f(2.3) 


e 


p water 


7j water 
Poise 


p corrected 
(2.2; 

Chmp = 0-05 
g cm -3 ) 


7] corrected 
(2.2'; 

Chmp = 0-05 
g cm -3 ) 


C 

(eau) 


C 

(Qhmp = 
0.05 

g cm" 3 ) 


for 

h= 10 cm 
r= 0.0001 
cm 

Qhmp = 
0.05 


(°C) (g cm" 3 ) 


(g cm -1 s" 


')(gcm- 3 ) 


(g cm" 1 s" 1 ) 


(cm" 1 s" 1 ; 


) (cm -1 s" 


1 ) 


15 


0.999126 


0.01139 


1.030598 


0.01381 


30640 


30038 


9 h 15 min 


16 


0.99897 


0.01109 


1.030438 


0.01345 


31472 


30853 


9 h 00 min 


17 


0.998802 


0.01081 


1.030264 


0.01311 


32290 


31656 


8 h46 min 


18 


0.998623 


0.01053 


1.030080 


0.01277 


33153 


32502 


8 h 33 min 


19 


0.998433 


0.01027 


1.029884 


0.01245 


33996 


33329 


8 h 20 min 


20 


0.998232 


0.01002 


1.029676 


0.01215 


34849 


34165 


8 h 08 min 


21 


0.998021 


0.009779 


1.029459 


0.01186 


35712 


35012 


7 h 56 min 


22 


0.997799 


0.009548 


1.029230 


0.01158 


36581 


35864 


7 h 45 min 


23 


0.997567 


0.009325 


1.028990 


0.01131 


37462 


36727 


7 h 34 min 


24 


0.997325 


0.009111 


1.028741 


0.01105 


38347 


37596 


7 h 23 min 


25 


0.997074 


0.008904 


1.28482 


0.01080 


39245 


38476 


7 h 13 min 



Particle size analysis is concerned with sedimentation of particles of 
different sizes. Some particles sediment more quickly than others and this 
results in variations in viscosity during the course of the experiment and 
also variations in the density of the fluid. Thus, in order to not diverge 
too much from the theoretical conditions established for mono-dispersed 
systems, a too significant concentration of the soil sample should be 
avoided (never higher than 1%). 



The graph of the sedimentation of a heterogeneous sample corresponds 
to a poly-dispersed system. This construction (Fig. 2.3) makes it possible 
to evaluate the percentage of particles with a diameter larger than a value 
"A" corresponding to a sedimentation time 't x \ 



26 



Mineralogical Analysis 



■d 

CD 

-i— > 

CD 

E 

TD 
CD 
C/) 

-i— » 

C 

o 
E 
< 



100% 




time 



Fig. 2.3. Curve of sedimentation of a complex sample (poly-dispersed system); 
t x is time representing the value of the sum of particles X. The 
intersection of the tangent at point X of the curve with the y-coordinate 
gives in A the quantity of particles larger than X 



2.2. Standard Methods 



2.2.1 Pretreatment of the Sample 
Adaptation to Soil Type 

The sample is dried at room temperature, sieved to 2 mm and carefully 
partitioned with a manual riffle sampler (Pansu et al. 2001). The weight 
of the test sample is 10 g; this quantity can be increased to 20 g for soils 
which are not very rich in clays. The treatments below are defined with 
respect to the different cases (A - H) listed in Table 2.3. 



Particle Size Analysis 



27 



Carbonated Soils (B) 

Reagents 
HCI 1 mol L" 1 



Table 2.3. Characteristic of soils subjected to analysis and preliminary treatments 



organo-mineral 
complex 



characterization 



treatment 



A: complex more or 
less saturated 



B: presence of 
carbonates 



C: rich in organic 
matter 

D: rich in organo- 
mineral cements and 
amorphous or 
individualised 
sesquioxides 

E: rich in sodic salts, 
no gypsum 



F: presence of Mn0 2 



G: presence of gypsum 
<10% 

H: presence of gypsum 
> 10% 



few drops of concentrated 
HCI do not cause any 
release of CO2, 
pH H 2 < 7 

HCI causes release of C0 2 
pH H 2 > 7 

OM content > 15% dried 
<2mm thin earth 

(Si0 2 + Al 2 3 ) > 10% of 

thin earth - ferrallitic, 
ferruginous tropical soils 



conductivity 1/5 > 0.3 mS 
cm -1 ; to 5 ml_ of aqueous 
extract, add 5 mL acetone 
- no precipitate 

strong reaction with H 2 2 ; 
violet colour of 
permanganate after 
oxidation 

positive reaction to acetone 
test 

estimation of gypsum by 
heating to 60°, then to 
105°C(cf. Sect. 2.2.4) 



treatment with H 2 2 at 1%, then 
30% 



perform two analyses: one without 
carbonate destruction, and one 
with destruction 

suitable treatment with H 2 2 



- dissolution of mineral cements 
with HCI and organic cements with 
H 2 2 

- treatment with Tamm reagent 

elimination of salts by washing 
and decantation-filtration 



pretreatment with bisulphite before 
destruction of organic matter 



elimination of gypsum by washing 
before agitation 

results difficult to interpret. Special 
techniques should be used 



28 Mineralogical Analysis 

Procedure 

The analytical standard X 31-107 (1983) recommends carrying out this 
treatment after destruction of organic matter with hydrogen peroxide 
(H 2 2 ). However, oxidation of the organic matter can temporarily 
produce oxalic acid which results in the neo-formation of calcium 
oxalate. It is thus preferable to eliminate the limestone at the pretreatment 
stage. 

- Put the sample (equivalent to 10 g soil dried at 105°C for example) 
weighed at ± 1 mg in a low form 1 ,000 mL Pyrex beaker. 

- Add 100 mL water. 

- Agitate and with a burette slowly add the diluted hydrochloric acid 
(1 mol L" 1 ) until complete destruction of the limestone present. 

- The pH should not go below 3.0. 

When all carbon dioxide is eliminated, boil gently for 5 min. Wash by 
decantation to eliminate calcium and excess acid (chloride test). 

Remarks 

- Avoid adding too much hydrochloric acid which can destroy the 
chlorites in certain trioctaedric chlorites. 

- Sodium acetate at pH 5 can be used to avoid attacking the lattice of 
certain clays. 

- In the presence of limestone particles involved in the grain-size 
distribution of the soil, an additional measurement will be required 
without destroying the CaC0 3 (Baize 2000). 

Destruction of Organic Matter 

Organic matter has a high aggregation capacity. It should thus be destroyed 
in the majority of soils (A - F in Table 2.3). Generally hydrogen peroxide 
at 30% (110 volumes) is used or stabilized hydrogen peroxide (Perhydrol or 
similar) in tropical climates. Some authors propose sodium hypochlorite or 
bromine in an alkaline medium (2 mol (KOH) L" 1 solution). 

Reagents 

- Pure hydrogen peroxide (30% - 1 10 volumes) 

- Ammonia (20%, d = 0.92) 

- Dispersant, 5% sodium hexametaphosphate solution 

- Sodium bisulphite NaHS0 3 

Case of Soils with Less Than 15% of Organic C 

Place the test specimen in a 1,000 mL Pyrex beaker. Add 100 mL of 1%. 
hydrogen peroxide. Leave in contact in the cold for one hour avoiding 



Particle Size Analysis 29 

excess foaming by agitating, or by using an aerosol to modify the surface 
charges (alcohol, etc.). 

Heat to 60°C and add a little 30% hydrogen peroxide to start the attack 
again. Add H 2 2 in small fractions until effervescence stops and there is 
discolouration of the supernatant. Bring to controlled boiling to destroy 
surplus H 2 2 and to reduce the volume without bringing to dry. 

Case of Soils Very Rich in Organic Matter (Histosols, Andosols, 
etc.) 

It is important to work on soils preserved in their natural moisture to 
avoid irreversible changes due to drying as these soils become 
hydrophobic. A sample equivalent to soil dried at 105°C should be used. 
The attack must be very gentle at the beginning because as soon as the 
oxidation reaction starts, it becomes violent; there is a sudden rise in 
temperature and a risk of overflow of foam. 

When sampling wet soil, add distilled water to form slurry. Add 50 mL 
hydrogen peroxide diluted to 1% and leave in contact in the cold. 

Heat each beaker to 60°C to start the reaction. If necessary, adjust the 
temperature by adding an ice cube made of deionized water. 

Add small fractions of hydrogen peroxide until there is no more foam, 
then bring to the boil. The liquid supernatant should be clear. Wash by 
decantation and continue the analysis. 

Remarks 

In certain cases, the organic matter may be "protected" by homogeneous 
mixture with carbonates and hydrogen peroxide then cannot act. If 
preliminary destruction of the carbonates (cf. "Carbonated Soils") is not 
sufficient, the organic matter should be attacked with hypobromite: 3 mL 
of bromine in 100 mL of frozen 2 mol (KOH) L 1 solution. 

Mix 50 mL of this mixture with the sample and wait 1 h; boil for 30 
min, let cool, add 200 mL distilled water; leave overnight, transfer on a 
filter and wash (three washings are sufficient) before dispersion. 

Presence of Mn0 2 (F) 

The presence of manganese salts can cause the rapid destruction of 
hydrogen peroxide. In this case the treatments should be renewed and the 
colour of the supernatant liquid monitored. Free manganese dioxide is 
soluble in hydrogen peroxide (Jackson 1969). 

Since manganese dioxide causes violent breaking up of hydrogen 
peroxide, it should first be reduced with sodium bisulphite (Petard, 1993). 
Before adding hydrogen peroxide, add 1 g of sodium bisulphite or 10 mL 
of an aqueous bisulfite solution at 37.5% to the sample. Add 50 mL 



30 Mineralogical Analysis 

deionized water and boil for 20 min to reduce the manganese dioxide in 
Mn 2+ ion. Then initiate the attack with hydrogen peroxide as described 
above. 

Presence of Amorphous Organo-Mineral Cements (D) 

Reagents 

- Tamm (1922) buffer: 10.92 g of oxalic acid + 16.11 g of ammonium 
oxalate for 1,000 mL; adjust to pH 3; 

- Hydrochloric acid 2 mol L" 1 solution: dissolve 166 mL concentrated 
HC1 (d= 1.18) in 800 mL water, agitate, cool and complete to 1 L. 

Procedure with Tamm Reagent 

Add 800 mL of Tamm reagent to a sample weighing 20 g; agitate cold, 
store in the dark for 4 h, centrifuge, and then filter. 

Procedure with HCI/H 2 2 Reagent 

Treat with 300 mL 2 mol (HO) L" 1 solution in a sand bath for 1 h at 
60°C. Elutriate and wash with deionized water. The acid solution and 
washing water should be collected and dried at 105°C. Weighing gives 
the mass M m of the soluble mineral fraction. If m represents the initial test 
specimen of soil, the mineral soluble fraction F m expressed as a 
percentage is calculated by F m = 100 M m /m. This value should be taken 
into account for the calculation of the balance of particle size 
fractionation. 

After dissolving mineral cements, organic cements should be dissolved 
as described in above "Destruction of Organic Matter". 

Presence of Gypsum (G and H) 

Procedure for Rough Estimate of Gypsum 

Put a test specimen of 10 g of soil sieved to 2 mm in an aluminium 

capsule. Place in a ventilated drying oven at 60°C for 24 h to eliminate 

the water of hydration. Cool in the desiccator and weigh = PI; place in a 

ventilated drying oven regulated at 105°C (for minimum 3 h) to eliminate 

the water of constitution; cool in the desiccator and weigh = P2. 

P1-P2 172 
Approximate percentage gypsum = 100 x ~ 50(,P1 -P2). 



Particle Size Analysis 31 

Procedure in Case of Gypsum < 10% (case G) 

At 25°C, gypsum is water soluble at a rate of 2 g L~ . After destruction of 
organic matter (cf. "Destruction of Organic Matter") and after destruction 
or not of limestone (cf. "Carbonated Soils"), put the sample (10 g) in a 
500 mL beaker with 300 mL distilled water on a magnetic stirrer with 
agitation. After 1 h, allow it to settle and elutriate the clear part, again add 
300 mL distilled water and repeat the operation (usually once again) until 
the acetone test is negative. 

For this test (cf. Chap. 30), use 5 mL aqueous extract, add 5 mL 
acetone, mix well; in the absence of gypsum, no precipitate will be 
formed. 

Procedure in Case of Gypsum >10% (case H) 

Determination of the exact particle size is difficult and it is advisable to 
adapt a specific method, for example, that of Vieillefon (1979). Complete 
dissolution of the gypsum enables the elementary composition of the non- 
gypseous part of the soil to be determined, but not of the total soil. 
Instrumental methods are more precise because only a short measurement 
time is required. Combined with desaturation using ion exchange resins, 
these methods make it possible to obtain satisfactory results before 
flocculation occurs. 



2.2.2 Particle Suspension and Dispersion 
Introduction 

Ions that can flocculate clay particles are ranked in descending order: 
Al 3+ > Ca 2+ > NH 4+ > K + > Na + > Li + 

The replacement of the natural compensation cations with high 
flocculating capacity is thus necessary to enable dispersion of the soil, i.e. 
the maintenance of the elementary particles in suspension. The stability 
of the suspensions is only obtained thanks to interactions between the 
diffuse layers of the same sign as clays and with control of the forces of 
attraction of van der Waals. 

Coulomb repulsion depends on the concentration of electrolytes and 
the valence of the ions. If the forces of van der Waals are higher than the 
force of Coulomb repulsion, the potential energy of the resulting 
interaction leads to flocculation. The aim is to chemically modify the 
distribution of charges and pH. 

For example, inversion of the edge charges can eliminate edge lattice 
attractions and produce negative particles with a weak attraction 
potential. For example hexametaphosphate is fixed by chemisorption on 



32 Mineralogical Analysis 

the octahedral cations present on the side faces of crystallites resulting in 
good dispersion. This dispersant is most often used when analyses are not 
required on the fractions after measurement of particle size. 

The pH should preferably be fixed at a value which is far from the 
zone of the isoelectric point (cf. Chap. 20) at which flocculation occurs. 
Generally it should be basic (approximately 10). A too high pH can 
induce solubilization phenomena. 

For oxides like Fe(OH) 3 , which can exist in a dispersed state with a 
positive or negative charge, adjustment of the pH to allow dispersion is a 
function of the isoelectric point. For Andisols, the pH for dispersion will 
often be acid (around pH 3.5) and always far from the isoelectric point. 

These treatments do not avoid lattice associations which are observed 
in kaolinites with a low negative charge or in micas (illites, muscovite, 
etc.). In this case it is necessary to use ultrasound for an additional 
mechanical effect. 1 The optimal amount of dispersant will depend on the 
effectiveness of the pre-treatments. Optimal effectiveness is obtained 
with an amount of sodium hexametaphosphate of between 20 and 50 
times the CEC. An excess of this reagent should be avoided as it causes 
flocculation while being adsorbed on colloids. For this reason, and when 
the fractions are needed for later analysis, it is better to use ion-exchange 
resins in Na + form as dispersants, e.g. Amberlite IR 120 Na (Rouiller 
et al.1972). These have the advantage of not causing any additional ionic 
charge of the medium, which is very favourable in the case of horizons 
with low clay content where a saline medium adds significant weight to 
the clay content. 

Among the dispersants that can be used are ammonia, soda, sodium 
carbonate, and sodium pyrophosphate. All these agents avoid 
compression of the double layer of crystallites and avoid raising the Zeta 



1 Ultrasounds: (see complementary bibliography for effects on soils). Ultrasonic 
vibrations are generated by magneto strictive oscillators. When a bar of a 
ferromagnetic material is subjected to a magnetic field, it changes length by 
magnetostriction. When this alternative field is applied in the axis of the bar, 
this causes an oscillation that is double of the applied field frequency. This 
vibration is transmitted to the suspended particles by the aqueous medium. 
The effect of cavitation with a frequency from 20 to 30 kHz makes it possible 
to break the forces of cohesion of the aggregates without causing significant 
damage to the elementary particles, as long as the application time is short. 
The treatment causes a rise in temperature which should be controlled. The 
apparatus are built so as to avoid the zones of resonance waves which are 
most destructive. Two types of apparatus are used, either with tanks or with 
probes with mechanical agitation by blades. Agitation with a bar magnet is 
not used except to recover magnetic particles if required. 



Particle Size Analysis 33 

potential, thus maintaining inter-particle forces of repulsion. It should be 
noted that at a rate of 17 mL per litre, ammonia does not cause an 
overload for weighing, and that soda dissolves the organic matter and 
precipitates iron, whereas pyro- and hexametaphosphate maintain it in 
solution. Aluminium is put in solution in the form of aluminates with 
soda, but not with ammonia. The responses are thus always slightly 
different. 

As the mixed hexametaphosphate-ammonia dispersant has one 
component with a higher density than water and one component with a 
lower density, the resulting density is close to that of water. 

Equipment and Reagents 

- Sedimentation cylinders, graduated 1,000 mL cylinders with ground 
stoppers (45/40 mm), 400 mm in length and 60 mm in diameter are 
generally used, but equivalent results can be obtained using transparent 
PVC tubes with an internal diameter of 71 mm and a length of 30 cm, 
with a filling mark at 1,000 mL, a square base, and stopped with a 
rubber stopper for agitation. 

- Dispersing solution. 15% sodium hexametaphosphate solution (calgon, 
(NaP0 3 ) 6 ) in deionized water. 

- 20% ammonia (d= 0.92) solution. 

- Ion exchange resin (Amberlite IR120 Na or similar). 

Procedure 

Treatment Using the Mixed Dispersant Hexametaphosphate - 
Ammonia 

After subjecting the soil sample to suitable pretreatments, quantitatively 
place it on an unfolded analytical filter and wash it with deionized water 
until dispersion begins. Pierce the filter and with jets of water from a 
washing bottle direct the soil into a cylinder (Fig. 2.4). Add 10 mL of 
15% sodium hexametaphosphate solution and 5 mL of 20% ammonia 
solution. Supplement with approximately 500 mL water, close and place 
on the rotary agitator for 2 h (4 h for clay soils). In the case of soils of 
andosol-histosol type, first carry out ultrasonic treatment for 15 min). 

After agitation, the sample should be well dispersed and its elementary 
particles (sands, silts, clays) quite separate from each other. After a few 
minutes, check there is no flocculation. Bring the volume to 1,000 mL 
with deionized water and homogenize. 



34 



Mineralogical Analysis 



Treatment Using Na Form IR 120 Resin 

Before treatment, the resin should be removed from particles smaller than 

200 |Lim by sieving. 

Place samples that have been subjected to suitable pretreatment 
quantitatively on a filter and wash with deionized water until dispersion 
begins. Pierce the filter and place the soil on a 200-|Lim mesh sieve in a 
funnel on a sedimentation cylinder to recover coarse sands. Wash, dry, 
and filter the recovered sands (cf. "Washing and Measuring Fine and 
Coarse Sands"). 

Place the fine particles in the cylinder. Add 50 mL wet Na form resin, 
then approximately 500 mL deionized water and agitate with the rotary 
agitator for 4 h (5 h for soils containing < 10% gypsum). 

After agitation, recover the resin on a 200 |Lim mesh sieve placed in a 
funnel in a 1,000 mL sedimentation cylinder, then wash separately and 
recover the washing water (the resin can be regenerated for another 
operation). 

The sample can also be brought to 1,000 mL directly with deionized 
water for sampling of the fine particles, which can be needed for later 
analyses. It is alsopossible to add 10 mL of 15% sodium hexametaphos - 
phate solution and 5 mL 20% ammonia, as in the procedure described in 
"Treatment Using the Mixed dispersant Hexametaphosphate - Ammonia", 
the densities and viscosities are then similar. 



Fig. 2. 4. Transfer in sedimentation cylinders for 
dispersion after pretreatment, filtration 
and washing 




Particle Size Analysis 35 

2.2.3 Pipette Method after Robinson-Kohn or Andreasen 
Principle 

The pipette method is based on sedimentation of the particles by gravity 
according to the law of Stokes (2.1). Recovery of the aliquot at a given 
depth and a given time makes it possible to identify a specific class of 
particles when all the particles bigger than the selected diameter have 
been eliminated. 

Equipment 

- Sand bath. 

- Robinson pipette on moveable frame with toothed rack (Fig. 2.5), 
aspiration by micropump with flow regulated at 60 mL min" 1 . The pipette 
should have undergone preliminary treatment to make it non-wetable (cf. 
"Hydrophobic Treatment of a Sampling Pipette"). 

- Fine aluminium capsules with a capacity of 3(MK) mL. 

- Drying oven with ventilation, regulated at 105°C. 

- Thermometer. 

- Balances, range: 120 g, sensitivity: 0.1 mg. 

- Sets of two sieves with 0.2 and 0.05 mm mesh, with a vibrating sieving 
machine. 

- Rotary agitator able to receive 10 or 20 cylinders, (30 rpm)- 

- Standard tributylchlorosilane. 

- Standard 1-chloronaphtalene. 

Preparation of Pipette 

Hydrophobic Treatment of a Sampling Pipette 

This treatment (Walker method) makes the walls of the pipette non- 
wetable and eliminates the need for rinsing between sampling. 

Prepare the smallest possible quantity of 4% solution of tributylchloro- 
silane in chloronaphtalene. Clean, carefully degrease the pipette, dry it 
well then treat the inside of the pipette by aspiration; drain, leave to dry 
for a minimum of 24 h at room temperature. 

Calibration of the Pipette - Overload Reagent 

This calibration should be done periodically. The same dispersing liquid 
used for the analyses is used again but the temperature should be checked 
as it influences viscosity. Put five fractions of 20 mL in tarred capsules, 
then weigh (± 1/10 mg). The average weight corresponds to the volume 



36 



Mineralogical Analysis 



removed. Dry for 5 h at maximum 105°C, weigh the capsules (± 1/10 mg) 
to determine overload due to the reagent. 

Procedure 

All operations should be carried out at 20°C in an air-conditioned room, 
and equipment and reagents should be kept at the same temperature. 

Control of Dispersion 

After agitation, lay the sedimentation cylinders out in line on the lab 
table, and open, taking care to include any deposits on the stopper. 



Sucti6n 



Evacuation 



u 



Andreasen 
Fig. 2.5. pipette 

Sampling 
system with 
the Robinson 

pipette (on the J — fl_ 
right) and the " 
Andreasen 
pipette (on the 
left) 



Pipette 
l/=20mL 



Two-way 
stopcock 



Two-way 
stopcock 



Soli 

suspension 



Robinson-K6hn 
pipette 



lxjwwix^a^wwi; * 



n 



10 cm 



Support 
with elevating 
rack 



First check the state of the suspension. Carefully check whether flocculation 
has occurred although this may be only partial and impossible to see. In 
case of doubt, complete the following steps: measure the temperature of 
the suspension, calculate (2.1) the falling time at 5 cm and 10 cm (for 
particles of 0.02 mm), hand shake the cylinder by turning it upside down 
and back for 1 min; start the chronometer, lower the pipette to 5 cm. Ten 



Particle Size Analysis 



37 



seconds before the time is up, remove a 10 mL sample and place it in a 
capsule. Lower the pipette to 10 cm and remove a sample at the 
corresponding time under the same conditions. Dry the capsules 
containing the samples in the drying oven at 105°C for 24 h, then weigh 
them (=t 1 mg); the two weights should be identical, any difference 
indicates flocculation and its extent. If any doubt remains about possible 
flocculation, it is better to start the analysis again. 

Certain apparatus make it possible to identify the optimal zone of 
dispersion for certain clays by bringing into play their property of 
orientation in an electric field. 

First Sampling (Clay + Silts) 

In the case of big series, a team of two people can be used, who should 
respect the timing very strictly (Table 2.4). Hand shake the cylinder 
containing the 1 ,000 mL suspension by turning it upside down and back 
to put all the deposits in suspension then place it on the counter top and 
start the chronometer (remove the stopper after the particles have been 
removed by agitation). 

- Close the three-way stopcock of the pipette; lower the pipette until 
the tip touches the surface of the suspension. Note the position of the 
index on the scale. Approximately 30 s before the time is up, (4 min 48 s 
at 20°C for 10 cm, see Table 2.5), carefully lower the pipette in the first 
agitated cylinder to the selected depth (here 10 cm). 

- Exactly 10 s before the sampling time, begin aspiration of 20 mL 
(speed intake 1 mL s" 1 ) by slowly opening the stopcock. The distribution 
around the exact sampling time gives the "average"'. When the liquid 
rises above the top of the stopcock, turn it off and run the overflow off 
through the side nozzle. 

Table 2.4. Procedure for stirring a series of sedimentation cylinders 



stirring first cylinder 




stirring 






1 min 


1 min 


second 
cylinder 
1 min 


1 min 


etc. 


when stirring is finished, start the 


lag 




lag 




chronometer: this is the 


time 




time 




beginning of the timed period 











Remove the pipette, quickly wipe the outside of the tube, and empty its 
contents into a previously tared 50 mL weighing bottle. Evaporate to dry 
and dry in the drying oven at maximum 105°C for 3 h. Weigh dry residue 
to precisely 1/10 mg. Correct the weight for overload due to the reagent. 



38 Mineralogical Analysis 

Remarks 

Aspiration should not be too fast to limit turbulence and to avoid 
aspirating particles with a larger diameter than those of the selected 
sampling range. Aspiration must also be regular. 

The pipette does not need to be rinsed between sampling since it has 
received hydrophobic treatment (cf. "Hydrophobic Treatment of a 
Sampling Pipette") and any error due to retention is negligible compared 
with other causes of error. 

Blank assays should be made with the dispersant alone (cf. 
"Calibration of the Pipette - Overload Reagent"). Weigh the sample and 
the blank after 24 h in the drying oven at 105°C. 

Second Sampling: Clay 

Table 2.5. right: Sampling time of particles (d = 2.65) by sedimentometry with a 
Robinson-Kohn pipette at a depth of 10 cm. 
left: Sampling depth of the clay-size fraction at different times 

clays < 2 jim tempe- clays < 5 |im < 20 |im < 50 |im 

rature <2 jam 



depth of sampling in cm T (°c) falling time at 1 cm 

after 

5h 6h 7h 8h 

6.2 7.5 8.8 10.0 20 8 h 00 1 h 16 h 04 min 47 s 

min min 48 s min 48 s 

6.4 7.7 9.,0 10.3 21 7 h 48 1 h 15 h 04 sedimenta- 

min min 00 s min 41 s tion 



6.5 7.9 9.2 10.5 22 7 h 37 1 h 13 h 04 

min min 12 s min 34 s 

6.7 8.1 9.4 10.8 23 7 h 26 1 h 11 h 04 

min min 30 s min 28 s 

6.9 8.3 9.7 11.0 24 7h16 1 h 09 h 04 

min min 54 s min 22 s 

7.0 8.5 9.9 11.3 25 7 h 06 1 h 08 h 04 

min min 18 s min 15 s 



method 



not 



possible 



Proceed as above after 8 h of sedimentation at 20°C (if necessary, a 
smaller depth can be used to sample clay on the same day: for example, 7 
h at 8.8 cm at 20°C - see Table 2.5). 

Weigh the residue exactly (± 1/10 mg) and correct the weight for 
overload due to the reagent (blank). 



Particle Size Analysis 



39 



Intermediate sampling can be performed; remember to take suspensions 
containing the coarsest phases first and the finest last. For this it is better 
to use an Andreasen pipette. 

Washing and Measuring Fine and Coarse Sands 

After the last sampling of clays, siphon off the supernatant liquid to 5 cm 
from the bottom; decant the deposit in 1,000 mL beakers. Add deionized 
water with a little hexametaphosphate (approximately 700-800 mL); 
agitate vigorously to put the deposit in suspension; after the time 
necessary for 0.02 mm particles to fall below the limit of aspiration of the 
siphon, siphon off the supernatant liquid (Fig. 2.6). 



5 cm 



25cml 
A 

5 cm 



Fig. 2.6. Washing of sands by decantation: left, first siphon off, right, continue 
siphoning until supernatant is clear (A = falling height of 0.02 mm 
particles) 

Again add water with a little hexametaphosphate; continue washing 
until the liquid supernatant is clear; finish with one final washing with 
distilled water; eliminate the maximum amount of water possible per 
decantation, quantitatively decant the deposit of sands in a capsule, put to 
dry in a ventilated drying oven at 105°C; after cooling, weigh total sands; 
put the sands on the top of two superimposed sieves, one with a 0.2 mm 
mesh (AFNOR 24), the other with a 0.05 mm mesh (AFNOR 18); sieving 
with a vibrating apparatus must be complete; check there are no cemented 
aggregates or plant debris. The mesh of the 0.2 mm sieve represents 
coarse sands, and the mesh of the 0.05 mm sieve represents fine sands. 

The coarse silts or very fine sands are determined by calculating the 
difference between total sands and the sum of coarse sands and fine 
sands. 



40 Mineralogical Analysis 

Remark 

The washing-decantation operations are long and tiresome, particularly 
as many samples are required in routine analyses. It is possible and 
advisable to use an automated system to wash the sands, e.g. the one 
developed by Susini (1978). 

Causes of Error 

A strict procedure is required to ensure the temperature remains stable for 
the duration of sedimentation. This can be achieved by immersing the 
cylinders in a thermostat bath, but this device is not really suitable for the 
treatment of the large series required in many laboratories. Consequently 
the cylinders are simply laid out in line on the lab table. The temperature 
should be taken in one of the cylinders at the beginning. Since the liquid 
medium presents good thermal inertia, variation is not very great over a 
short period, i.e. for the first sampling of about 4-5 min. The most 
favourable temperature is between 15°C and 25°C; above 30°C there is a 
risk of flocculation; below 15°C the times needed for sedimentation will 
be too long. For these reasons, in the absence of a thermostat, it is best to 
work in an air-conditioned room. 
But the main causes of error are: 

- Too abrupt entry of the pipette in the suspension. 

- Error in the depth of sampling. 

- Irregular or too rapid aspiration; certain authors recommend a time of 
20 s per aspiration for a volume of 10 mL. These requirements exclude 
aspiration with the mouth. 

Optimal working conditions are guaranteed by making sure there is no 
variation between the way the operators perform the series of operations, 
i.e. moving the pipette, lowering the pipette into the suspension, exact 
timing of the beginning of the sampling, very regular sampling, exact 
volume, careful removal of the pipette, draining of the sample into the 
capsule, next sampling. The ideal solution is to use a simple automatic 
unit like that described in Pansu et al. (2001). However, even if the 
traditional manual system has to be used it is preferable to carry out 
aspiration with a small electric pump with a fixed flow; peristaltic pumps 
fulfil this function well. 



Particle Size Analysis 41 

Calculations 

Collected Data 

Mass of soil sample (air dried) = m 

Moisture correction coefficient = 

mass sample after drying at 105°C lm= K 

Volume of the sample = V v 

Mass of blank (reagents without sample) after drying 105°C = m B 

Mass of first sample (clay + silt) after drying 105°C = m x 

Mass of second sample (clay) after drying 105°C = m 2 

Mass of total sands after drying 105°C = m 3 

Mass of coarse sands (rejected by 0.2 mm sieve) 

after drying at 105°C = m 4 

Mass of fine sands (rejected by 0.05 mm sieve) 

after drying at 105°C = m 5 

Calculation of the Results in% of Soil Dried at 105°C 

Clays = C = (m 2 - m B ) x 1000 x 100 / (V p x m x K) 

Silts = Si = (mi -m 2 x 1000 x 100 / (V v x m x K) 

Fine sands = FS = 100 x m 5 1 (m x K) 

Coarse sands = CS = 100 x m 4 / (m x K) 

Total sands = S= 100 x m 3 / ( m x K) 

Coarse silts = CSi S-(FS + CS) 

If limestone is present (Table 2.3, B), and particle size analysis was 
performed without destruction of carbonates, carbonates should be 
determined on the separated fractions which provides information about 
the distribution of limestone. 

Checking and Correction of the Results 

Taking into account the moisture correction factor (cf. "Calculations" 
under Sect. 2.2.3), the sum: clays + silts + total sands + organic matter + 
if necessary, carbonates, soluble salts, gypsum, must be between 95 and 
102%, preferably between 98 and 102%. Soils rich in organic matter can 
provide too high balances: in the event of incomplete destruction during 
pretreatment, organic matter can be counted twice. 

A too small sum results from losses during the pretreatments. In the 
majority of cases, it is impossible to determine the exact proportions of 
losses in organic matter, soluble salts, carbonates and gypsum. An overall 
estimate of the losses can be made as follows: using the cylinder in which 
the samplings were made, add 10 mL of 1 mol (CaCl 2 ) L" 1 solution and 1 
mL of 1 mol (HO) L" 1 to flocculate colloids and prevent the formation of 



42 Mineralogical Analysis 

calcium carbonate during drying in the drying oven. Allow the particles 
to deposit, completely remove the clear solution, put the deposit in a tared 
capsule, dry in the drying oven at 105°C, and then weigh. This gives 
mass m r from which losses during the treatments (organic matter, etc.) 
can be determined and the balances corrected. 



2.2.4 Density Method with Variable Depth 
Principle 

This type of analysis is advantageous because it avoids fractionation of 
the sediment in dimensional classes and allows the construction of curves 
of distribution. In the density method proposed by Bouyoucos (1927, 
1935, 1962), the heterogeneous suspension is considered to behave in the 
same way as a homogeneous liquid with the same density. Casagrande 
(1934) showed that it is then acceptable to use a float densimeter to 
measure the average density in the suspension column with the float. The 
plan of average density is located at a distance H R from the highest level 
of the suspension. From (9.4) and (9.4') it separates the particles from the 
radius: 

(t = sedimentation time), and allows particle size analysis. 

The density method can be performed with permanent immersion, 
which fulfils the conditions of continuous measurement, or by temporary 
immersion, which resembles discontinuous measurement described in 
Chap. 1, without the same precision, but for routine measurements it 
has the advantage of avoiding sampling and weighing. On the other hand, 
the density method requires a larger number of samples than the pipette 
method. 

Equipment and Reagents 

- Cylinders identical to those described in "Equipment" under Sect. 2.2.3, 
special conical hydrometers to avoid accumulation of the particles on 
the surface, thermometer, pycnometers, magnifier with long effective 
focal spot. 

- Dispersing reagents of "Equipment and Reagents", isoamylic alcohol. 



Particle Size Analysis 43 

Checking the Hydrometer 

Calculation of Falling Height of the Particles 

The relation of Casagrande makes it possible to calculate the value H r 

giving the effective depth selected as falling height of the particles. For 
this calculation, the measurements shown on the hydrometer in Fig. 2.7 
are required. The volume of the hydrometer is obtained by liquid 
displacement. 

H r = h x + Q.5{h-V/S) (2.5) 

V: volume in cm 3 
S: section in cm 2 
A graph can be drawn representing depths H r as a function of the 
densities. This makes it possible to draw up the table giving the size of 
the particles as a function of temperature, time, and depth, for an 
unknown hydrometer. 

Checking the Graduations on the Hydrometer 

This test is made with pure water and a 2% solution of barium nitrate or 
chloride. Use a pycnometer to measure the density of water (note the 
temperature) then the density of the 2% solution (4 significant decimals). 

Take the same measurements by submerging the hydrometer in the 
test-tube successively containing the two liquids; read the densities with 
the help of a magnifier with a long focal spot (5 or 6 cm). 

Note the differences in the measurements obtained with the 
pycnometer and the hydrometer. If the relative values are the same, the 
hydrometer is valid even if the indications are not exact, because in the 
calculations the differences in density are used. 

If the differences between the pycnometer and hydrometer measurements 
are not constant, an abacus of transposition has to be established: values 
read on the hydrometer/actual values (this is very seldom the case). 

This method is similar to that described in Sect. 2.2.3 using 30 g of 
fine earth (soil air dried and sieved to 2 mm). For dispersion, add 30 mL 
of 102 g L" 1 hexametaphosphate solution. Agitate for 4 h by upside 
down and back rotary shaker, transfer in the cylinders and complete to 
1,000 mL. 



44 



Mineralogical Analysis 



H, 



h 



h/2 



0.995 



Density 
scale 



1.020 





1.02- 

1.015- 

£ 1.01 ■ 

8 1005- 

D 1 : 

ft QQ^ ■ 


















































u.yyo 
0.99 










j 10 15 20 25 
H r {cm) 



Fig. 2.7. Relation between density and falling height of the particles H r (2.5) for a 
hydrometer with the following characteristics: V= 45 cm 3 , S = 28.26 cm 2 , 
/? = 17 cm, /?-, = 15.2 cm). 



Procedure 



Preparation of the Sample 



Fig. 2.8. Positioning of hydrometer 




Guiding 
paper 



Particle Size Analysis 



45 



Measurement of the Clay + Silt (0.02 mm) Fraction 

Table 2.6. Sizes of the particles as a function of temperature, time and the depth: 
valid for a standard hydrometer (laboratory of sedimentary sequences, 
IRD Bondy, France, unpublished data) 



Particle 


t 


density^ 


1.020 


1.015 


1.010 


1.005 


1.000 


Size 


(°C) 


(min -I) 


8.7 cm 


11.6 cm 


14.4 cm 


17.2 cm 


20.1 cm 






4 


21 Mm 


24.7 Mm 


27.4 Mm 


30.1 Mm 


32.5 Mm 




15 


6 


17.4 


20.1 


22.4 


24.5 


26.5 






8 


15 


17.4 


19.4 


21 


23 






9 


14.2 


16.4 


18.2 


20 


21.6 


20 Mm 




4 


20 


23 


25.8 


28.2 


30.3 






6 


16.4 


19 


21 


23 


24.5 




20 


8 


14.2 


16.3 


18.2 


19.8 


21.4 






9 


13.4 


15.4 


17.2 


18.8 


20.2 


(clays + 




3 


21.8 


25.2 


28.1 


30.7 


33.2 


silts) 


25 


4 


19.9 


21.8 


24.3 


26.6 


28.7 






6 


15.4 


17.8 


19.8 


21.6 


23.4 






8 


13,4 


15,4 


17,2 


18,8 


20,3 


particle 


t 


density^ 


1.020 


1.015 


1.010 


1.005 


1.000 


size 


(°C) 


(hours i) 
















6 


2.2 Mm 


2.6 Mm 


2.9 Mm 


3.1 Mm 


3.4 Mm 




15 


8 


1.9 


2.2 


2.5 


2.7 


2.9 


2 Mm 




24 


1.1 


1.3 


1.4 


1.58 


1.7 






6 


2.1 


2.4 


2.7 


2.9 


3.2 




20 


8 


1.8 


2.1 


2.3 


2.5 


2.7 


(clays) 




24 


1 


1.2 


1.4 


1.5 


1.6 






6 


2 


2.3 


2.6 


2.8 


3 




25 


8 


1.4 


2 


2.2 


2.4 


2.6 






24 


1 


1.1 


1.2 


1.4 


1.5 



If possible measurements should be made in an air-conditioned room at a 
constant temperature of 20°C. The cylinders containing the suspensions 
are grouped; check their volume has been completed to 1,000 mL, 
measure the temperature by referring to a table (such as Table 2.6) giving 



46 



Mineralogical Analysis 



times of sedimentation for measurements at the selected temperature (for 
example 4-6-8-9 minutes). 

Hand shake the first cylinder by it turning upside down and back for 
1 min, add an isoamyl alcohol drop anti-foamer, start timing, introduce 
the hydrometer very gently so as not to disturb the suspension (this is the 
most delicate part and a significant cause of error), take care that the 
hydrometer is maintained in the centre of the suspension, (if need be, 
make a paper guide see Fig. 2.8), take the readings at the top of the 
meniscus at 4-6-8 and 9 min. Continue in the same way with the 
following cylinder. 

Take a reading of the blank in a cylinder containing only the 
dispersant. 

Measurement of Clay Fraction (< 0.002 mm) 

The time counted starts at the beginning of agitation of the first cylinder 
during the first reading (clay + silt). To define this time, the average 
temperature has to be calculated from the beginning of sedimentation 
until the reading; take readings with the hydrometer at 6-8-24 h carefully 
respecting the 2 min interval used for the first sampling; transfer the 
hydrometer very carefully from one cylinder to another in order to avoid 
disturbances. 



Fig.2.9. Example of abacus: 
sizes of particles are a 
function of the time of 
sedimentation for a 
given hydrometer and 
a density close to 
1.010. 



on 


1.27 | 

S 25 -: 

-1—' 
E 23 ": 

■% 21-: 

% 19-: 
B 17" 

°- 1«i- E 


\ 






































* 


V 












































I o 


\ 5 6 7 8 9 10 
Sedimentation time min 



Remarks 

As can be seen in Table 2.6, that selected times do not correspond exactly 
to the selected particle size; however, in a small space of time, variation 
in particle size is considered to be continuous; this makes it possible to 
plot an abacus around a given depth (Fig. 2.9) which then makes it 
possible to define an exact falling time for a given particle size. 



Particle Size Analysis 47 

Calculations 

The percentage of particles P corresponding to a given density is 
obtained from the equation: 

100 Ps ll \ \ 

P = 1 \ [[P ±S P)-Pf) v - ( 2 ' 6 ) 

™\PS-Pf) 

p s = density of solid = 2.65 for soil; 

P/= density of the dispersing solution; in our conditions at t°C, one can 

calculate p f by means of (2.2); 

p = density read at t°C; 

S p = corrections + or - on readings to bring them to 20°C (Table 2.6); 

V= volume, 1,000 mL; 

m = soil sample, 30 g; 

The calculation can be simplified by calculating K = 100 p s I m (p s - pf) 

which gives P% =K[(p± S p )- p f ]V 

with m = 30 g, at 20°, it gives K = 5.35 

Determination of Sands 

Use the same technique as that described in Sect. 2.2.3. 

2.2.5 Density Method with Constant Depth 

The density method with variable depth (cf. 2.2.4) has the advantage of 
greater speed compared to the pipette method as well as allowing 
uninterrupted measurements if required. However, as the depth of 
immersion is not constant, the degree of precision is lower and 
calculations are longer. 

The chain hydrometer (de Leenheer system) makes it possible to 
measure the density of the suspension to a given constant depth of 
approximately 20 cm, which in turn, makes it possible to approach the 
principle and the precision of the pipette method while avoiding sampling 
and weighing (De Leenheer and Macs 1952; De Leenheer et al. 1955; 
De Leenheer and van Hove 1956; van Ruymbeke and de Leenheer, 1954). 

The apparatus (Fig. 2.10) is composed of an immersion body at the end 
of an arm with (1) a pointer that identifies the level of the liquid and (2) 
at the top, a support that can receive overload weights in the form of 
riders, and an equilibrium chain that allows a very fine fit when the 
pointer locates the surface of the liquid. The depth of sedimentation is 
represented by the distance from the point of the needle located in the 
middle of the body of the hydrometer. 



48 



Mineralogical Analysis 



Rider 



* 





v 




i 




I ■ 






1 






* j—* Equilibrium 


Pointer 


v > x chain 


of level **%,> 


x - 


^Y 


* 




I 






of f 

sedimentation jr 


™ t\ 


* 
X 


Fig. 2.10. 




— 


>■ 


Principle of 






x 


chain 




>: 


hydrometer 




X 


after De 




X 


Leenheer 




K 


and van 




>: 


Hove(1956). 






^ 




>: 



One minute before measuring time, carefully introduce the hydrometer 
into the suspension; add the weights in such way that the reference mark 
of the needle is 1 - 2 cm above the level of the liquid. At the precise time 
of the sampling, adjust the needle so that it is in contact with the liquid by 
quickly adjusting the chain with the screw device. The chain can cause an 
overload of 100 mg. The reading for total overload gives the weight of 
the hydrometer. Having determined in advance the volume of the 
hydrometer (by immersion in distilled water), one thus obtains: 
density (p) = weight of the hydrometer / hydrometer volume 
Continue the calculations in the same way as for the density method 
with variable depth (2.6). 

2.2.6 Particle Size Analysis of Sands Only 

Place 100 g of fine earth (standard 2 mm preparation from a perfectly 
homogenized batch) in a 1,000 mL beaker with 100 mL hydrogen 
peroxide brought to 30 volumes; leave to act in a cold place overnight, 
then transfer on a moderately heated hotplate; add hydrogen peroxide in 
small fractions until complete destruction of the organic matter, then 
eliminate excess hydrogen peroxide by boiling without going to dry. 



Particle Size Analysis 



49 



Table 2.7. The two sieving columns used successively for particle size analysis of 
sands 





column 1 




column 2 


mm 


AFNOR 


mm 


AFNOR 




standard 




standard 


2 


34 


0.315 


26 


1.6 


33 


0.25 


25 


1.25 


32 


0.20 


24 


1 


31 


0.16 


23 


0.8 


30 


0.125 


22 


0.63 


29 


0.100 


21 


0.50 


28 


0.08 


20 


0.40 


27 


0.063 


19 






0.050 


18 



Transfer the residue in a cylinder and add 500 mL distilled water and 
25 mL of 52 g L" 1 sodium hexametaphosphate solution. Place in a 
rotating shaker for 4 h in the same way as for complete particle size 
analysis. 

After this operation, transfer the content of the cylinder on a 0.05 mm 
mesh sieve (French standard AFNOR NF-X- 11-504 module 18); wash 
the residue under running water. 

Place the well-washed sands_of the 0.05 mm sieve in a 250 mL beaker. 
Add 100 mL of 6 mol (HO) L" 1 solution, cover with a beaker cover, and 
boil gently for 2 h to dissolve iron. 

After cooling, decant and wash by successive decantation until 
complete elimination of the acid; transfer again on a 0.05 mm sieve, 
wash, and transfer quantitatively the sands in a capsule; dry for 24 h in 
drying oven at 105°C. Let cool and weigh total sands. 

Sieve dry total sands successively on the two columns of sieves 
(Table 2.7). Place the columns successively on a vibrating sieve machine 
(Pansuetal., 2001). 

Transfer in column 2 the fraction collected at the bottom of column 1 . 
Sieve 10 min and weigh each fraction (± 0.01 g). 

Checking: sum of weighings of each fraction = total sands. 



50 Mineralogical Analysis 

2.3. Automated Equipment 

2.3.1 Introduction 

The phenomena of slaking of the soils requires precise knowledge of the 
grain-size distribution of the 2-20 |Lim fraction; sediment studies require a 
distribution of the fine phases down to 0.1 |Lim or even lower. In 
agronomy, the horizons comprising the formation of clay are studied 
using ratios for "coarse clay < 2 |Lim/fine clay < 0.2 |Lim". Since gravity 
methods cannot provide all the answers, a range of different techniques is 
required. 

Table 2.1 summarizes the main methods used for measurement of the 
particle-size distribution of soils. Some methods are well suited for the 
repetitive measurements needed for studies in the fields of pedology, 
agronomy, geology or sedimentology; others are more suitable for 
detailed and in-depth studies. The choice of a method will depend on: 

- the degree of precision required, reproducibility and repeatability, and 
a good correlation with the pipette reference method (cf. 2.2.3) despite 
its defects; 

- the extent of the particle size field and possibility of extending it to 
sub-micronic or nanometric particles; 

- the speed of execution, the time needed to produce a result, the 
flexibility of use, the possibility to significantly increase the number of 
analyses; 

- the possibility of recovering the particle fractions for later 
measurements, or of taking other measurements simultaneously 
(continuous analyses, etc.); 

- the cost of equipment and personnel, the importance of the request and 
available space; 

- continuous data acquisition and exploitation (monitoring, calculations, 
histograms and cumulative frequency curves). 

Given the wide granular spectrum of soils, combinations of individual 
methods that do not cover the complete spectrum are often used. There 
should be a significant overlap between the methods. The pipette method 
remains the reference method for all comparisons; analysis by laser 
diffraction makes it possible to extend the spectrum to the sub-micronic 
field but still identify silts. The true representativeness of equivalent 
diameters in a given class can be checked using a microscopic method 
and image analysis. 



Particle Size Analysis 



51 



2.3.2 Methods Using Sedimentation by Simple Gravity 

The majority of apparatus designed to mechanize the pipette method are 
not widely distributed, as each laboratory tends to develop devices 
suitable for its own needs. For example, the automatic particle- 
measurement instrument distributed by "Technology Diffusion France" 
(Pansu et al., 2001) is based on sedimentation in a thermostated cupboard 
with an automated pipette. 

Analysts, especially those in the industrial sector, need methods that 
obtain rapid results with good repeatability and the possibility of 
calibrating the apparatus (using calibrated microball powders). For the 
main types of automated equipment available and the names of 
manufacturers see Pansu et al. (2001). 

Sedimentation Balances and Automated Sieve Machines for 
Wet Measurements 

These balances (Sartorius, Cahn, Mettler, etc.) make it possible to 
continuously record the process of sedimentation between approximately 
1 and 150 |Lim (Fig. 2.1 1), the higher fractions being in the domain of auto- 
mated wet or dry sieve test machines (Micromeritics, Seishin, etc.). 



Fig. 2.11. 



Diagram of a 
particle size 
balance with 
constant 
equilibration 




=FT 



/ 



Detection 

system 

of equilibrium 



Command 
card 



c 



: 



Motor 

for 

equilibration 



Sedimentation is carried out after dispersion on samples of reduced 
weight, i.e. about 1-2 g, in a thermostatic enclosure. Continuous 
automatic recording of the weight of the sediments deposited makes it 
possible to create cumulative mass curves as a function of time. 
Depending on the degree of automation and calculation, frequency charts 
can also be created. The measurements are reproducible, but require a 
long time to perform and are thus not suitable for series analysis. 



52 Mineralogical Analysis 

Systems Using Simple Gravity and Measurement by X-ray 

The particles are prepared and put in suspension (as described in sections 
X-ray beam). The particles absorb a quantity of X-ray proportional to 
their number. The resulting intensity is measured by a scintillation 
counter. 

At the beginning, the resulting intensity of X-rays is at a minimum, 
then the falling particles cause an increase in the intensity transmitted. To 
reduce measurement time, the cell containing the suspension gradually 
moves downwards and the fixed X-ray beam sweeps a portion of the 
suspension increasingly close to the surface. All these movements are 
controlled by computer, and the position of the cell is a logarithmic 
function of time, coupled with the x-axis of the recorder, which makes it 
possible to determine the diameter that corresponds to the position of the 
cell. The smallest diameter it is possible to measure is 0.1 |im and the 
largest is 100 - 300 |Lim, depending on the model. 

Continuous measurements make it possible to express the results in the 
form of cumulative curves of histograms of weight, or number of surface 
particles, etc. Computer interfaces make it possible to store the results of 
the analyses, and a sampler equipped with a carrousel allows 
uninterrupted treatment of several samples. 

It should be noted that X-rays with wavelengths of less than 10 nm are 
well suited for the measurement of particles which would be impossible 
to measure in visible light (100 - 800 nm i.e. similar to the diameter of 
fine clay particles). Repeatability is satisfactory and measurement up to 2 
|Lim takes about 10 min. 

This type of equipment has been the subject of comparative studies 
with the pipette method for the analyses of soils (e.g. Delaune et al., 
1991). It is useful for the 50-1 |um fraction, but needs a longer time for 
finer particles (< 0.2 |Lim in 50 min). However, undervaluation of coarse 
silts has been observed when they comprise more than 20% of the soil. 

System Using Simple Gravity and Measurement by Light 
Absorption or Scattering 

Methods based on photo-sedimentation (nephelometry, turbidimetry) are 
subject to many interferences. Their reproducibility and repeatability are 
low due to the use of white light or monochromatic light in the visible 
spectrum. 

These methods should only be used for rapid comparisons within 
homogeneous families. 



Particle Size Analysis 53 

Methods Using Elutriation 

In these methods, the falling speed of the particles (the mobile solid phase 
which is easy to measure) is lower, equal or higher than the speed of the 
fluid (non-stationary mobile liquid phase). 

The liquid phase circulates in the reverse direction to the particles, 
making it possible to sort them; the finest particles migrate upwards or 
fall by gravity. The different fractions can be recovered. This system is 
suitable for certain studies on sediments, but measurements take a long 
time and are not really suitable for repetitive analysis. 



2.3.3 Methods Using Accelerated Sedimentation 
Principle 

In practice, methods using simple gravity cannot be used for particle sizes 
<2 |Lim because of the extremely long time needed for sedimentation of the 
finest particles. It is difficult to maintain the cylinders of sedimentation 
without convection currents for long periods and to withdraw the particles 
from the Brownian movement. However, acceleration of gravity by 
centrifugation makes it possible to exceed the limits and to mitigate the 
effect of Brownian movement. 

The techniques of separation and the recovery of granulometric phases 
by this process are discussed in Chap. 3. 

Apparatus Using Centrifugal Discs 

Some equipment uses successively first simple gravity with the vertical 
rotor remaining stationary for the largest particles, and second gravity 
with centrifugation at speeds of 1,800 to 8,000g (Horiba, Shimadzu, 
Seishin, Union-Giken, Joyce-Loebl-Vickers, etc.). 

Analysis is continuous, and recording makes it possible to 
automatically create curves and histograms. Masses of soil of the order of 
1 g can be treated in this way. 

Depending on the manufacturer, the measuring cells are intersected 
either by a filtered incandescent light with measurement of absorption, or 
- very exceptionally - by laser or X-ray detection (Brook Haven). 
Particles of 0.01 |Lim to 100 |Lim can be identified. Other manufacturers 
use horizontal discs and samplings at a given distance and at a given 
time. The fractions are dried, weighed and possibly subjected to other 
analyses (Fritsch, Simcar, Joyce-Loebl, etc.). 



54 Mineralogical Analysis 

The performances of these apparatus are not always equal for series 
analysis, and repeatability is not always within the range usually obtained 
with the pipette method. 

For sub-micrometric analysis, one manufacturer offers an ultracentriflige 
with a titanium disc at 100,000g in a partial vacuum to avoid heating and 
noise. A UV scanner (280 nm) makes it possible to analyze soil particles of 
less than 500 nm (Beckmann Spinco). 

Micro-methods using Field flow fractionation (FFF) are still not 
reliable enough for widespread use in soil studies. 



2.3.4 Methods Using Laser Scattering and Diffraction 

Laser particle-measurement instruments have undergone spectacular 
development and can now be used for an increasing range of particle 
sizes. Certain equipment make it possible to cover ranges from 0.1 to 
2,000 |Lim, but in general, apparatus are particularly powerful for a more 
limited range. One range is dedicated to sub-micronic, or even 
nanometric particles, while others with a wider range are particularly 
useful for the particle size analysis most usually required by soil 
laboratories. 

These measurements are not based on sedimentation and must 
consequently be calibrated. 

A dispersing liquid containing suspended particles circulates in a 
measuring cell intersected by a monochromatic Laser beam collimated by 
a condenser on a window of analysis of a defined surface. The light of the 
Laser is diffracted on the outside of the particles and the angles of 
diffraction are inversely proportional to the size of the particles. An 
optical system collects the signals which are analyzed by Fourier 
transformation and discriminated on a detector engraved with pre- 
determined angles. The signal is treated to extract the distribution of the 
particles. The results can be expressed in the form of curves: by average 
diameter (particle size distribution) expressed as a percentage of total 
weights, by histograms of weight, surface, number of particles, volume, 
etc. 32 - 64 classes of sizes can be measured (Malvern, Cilas-Alcatel, 
Coulter, etc.). 

Certain apparatus allow either proportioning on a suspension, or on dry 
powder, which can be useful for analyzing silts. Serial deflocculation on 
line is possible by ultrasound. Loading 40 samples with a sample 
distributor and a using distributor for reagents makes it possible to work 
without continuous monitoring. 

Analytical files enable methods to be pre-determined, including the 
dispersants. The procedures are simple but vary considerably with the 



Particle Size Analysis 55 

apparatus and it is consequently impossible to give a detailed procedure 
here. 



2.3.5 Methods Using Optical and Electric Properties 

Analysis of the distribution of sub-micron particles (3 nm to 3 |im) 
combines measurements of pH, temperature, conductivity, and relative 
viscosity making it possible to control the stability of a suspension and 
the electro-kinetic potential (Zeta potential, potential difference between 
the dispersed surface layer and the medium of dispersion). 

The ionic force is measured in an electrophoresis quartz tank with a 
Pt-Mo electrode on particles measuring from 1 to 1,000 |Lim (Malvern, 
Brookhaven, Coulter, Micromeritics, Mono-Research Lab., Zetameter 
Inc., Matec Applied Science, etc.). 

The effective surface charge of the particles is determined by the 
measurement of mobility in a liquid-solid system, the permittivity of the 
liquid being known. This enables the study of the phenomena of 
flocculation and dispersion. 

Other apparatus are designed for the study and optimization of the 
dispersion of certain clays for industrial use. They make it possible to 
differentiate flocculated and deflocculated particles. For example, 
primary Kaolinite particles consist of regular hexagonal discs. The nature 
of these particles means that in the presence of an electric field a dipole is 
induced, causing alignment with the field. The neo-aggregates formed by 
flocculation consist of clusters of randomly arranged primary particles, 
out of alignment with the electric field. To measure the relative 
proportions of flocculated and dispersed particles, the suspension is 
intersected by a Laser beam and the diffused light is analyzed. The result 
is quantified. Measurements made in different conditions (dispersing 
concentration, the nature of the dispersant, pH, etc.) enable optimization 
of the analyses. 



2.3.6 Methods Allowing Direct Observation of the Particles 



Optical and Electronic Microscopy - Radiation Counter and 
Image Analyzer 

These direct methods are based on the use of optical microscopy, or 
possibly of electronic microscopy (cf. Chap. 8). Particle fractions isolated 
by gravity can be used among others. In electronic microscopy, the 
preparations must be dried and presented on grids or plates (MET-MEB). 



56 Mineralogical Analysis 

Microscopy makes it possible to directly observe the population of 
particles and their morphological parameters. Shape, length, width, 
thickness or diameter can be defined on micro-samples, and the fractal 
properties estimated. Comparison of the particles of the same fraction 
makes it possible to judge the quality of fractionation (use of tests of 
bulky diatoms, regularity, influence of density, etc.). Counting can be 
done by a radiation counter, or by an image and texture analyzer. 

In electronic microscopy with an EDX probe it is possible to perform 
chemical analyses; and in optical microscopy, to use infra-red radiation. 
This equipment offers a wide range of possibilities (Quantachrome, Zeiss, 
Leitz, etc.) and makes it possible to establish percentages of cumulated 
mass, distribution by size of particles, in terms of number, mass and 
specific surface area (0.1 - 300 |Lim). 

2.3.7 Methods Using Conductivity 

While passing through a gauged opening, a particle displaces a volume of 
electrolyte which modifies electric resistance (differential conductivity). 
This change in resistance is a function of volume. Counting allows 
particles to be grouped in classes using an amplitude discriminator. 

The results are expressed in 16 counter channels as total percentage 
weight or the number of particles of a specific dimension; results can be 
presented in the form of graphs or tables. 

The apparatus based on this principle are counters that make it possible 
to ignore density which can be useful when dealing with soils rich in iron 
oxides with a high density (4 - 5), i.e. well above the mean of 2.65 used 
for sedimentation by simple gravity. The shape of the particles is 
significant for the accuracy of the measurements. Measurements are 
possible between 1 and 5,000 |Lim (Coulter), but the optimal field of 
measurement depends on the choice of a suitable opening. Particles of 
less than 1 |Lim are often underestimated. The preparation of the samples 
is identical to the standard method (cf. Sects. 2.2.1 and 2.2.2). 



References 

Atterberg A (1912) Die mechanische Bodenanalyse und die klassifikation der 

mineral boden sechwedens. Int. Mitt. Bodenk.,2, 312-342 
Baize D (2000) Guide des analyses courantes en pedologie., INRA, France, 257 P 
Bouyoucos GS (1927) The hydrometer as a new method for mechanical analysis 
of soils. J. Soil Set, 23,343 



Particle Size Analysis 57 

Bouyoucos GS (1935) A hydrometer method for making mechanical analysis of 

soils. Bull. Am. Ceram. Soc, 14, 259 
Bouyoucos GS (1962) Hydrometer method improved for making particle size 

analysis of soils. Agron. J., 54, 464-465 
Casagrande A (1934) Die Araometer-methode zur Bestimmun der kornverteilung 

von Boden und anderen materialen. Springer J. 
De Leenheer L and Van Hove J (1956) Werkwijze voor de mechanische analyse 

met de kettinghydrometer. Rijksland Bouwhogeschool (Gand)., XXI, 

249-274 
De Leenheer L and Maes L (1952) Analyse granulometrique avec l'hydrometre 

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58 Mineralogical Analysis 

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60 Mineralogical Analysis 

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Particle Size Analysis 61 

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62 Mineralogical Analysis 

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Instrumental Methods 

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Particle Size Analysis 63 

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Fractionation of the Colloidal Systems 



3.1 Introduction 

The identification and the quantification of the finest soil fractions are 
essential to explain the transformation of minerals, as these fractions are 
directly related to pedogenesis and, in agronomy, to potential fertility. 

The nature and properties of these particles are of interest to agrono- 
mists (soil chemistry and physics: textural class, fertility, pore system, 
water storage, cohesion, slaking, etc.), soil scientists (pedogenesis, chara- 
cterization and functioning of soils, lithological nature, products of 
alteration, etc.), geologists of the quartz period (sedimentology: origin of 
wind, marine, or lake deposits, typology of volcanic ash and heavy 
minerals, etc.), mineralogists and geochemists (assessments of alterations, 
mineral stocks liable to alteration, origin and nature of materials, etc.). 

The majority of the instrumental methods used for the determination of 
the texture of the soils (cf. Chap. 2) do not enable isolation of the fractions 
measured, but discontinuous methods based on sedimentation do enable re- 
use of the sand, silt and clay fractions, as long no contaminating 
dispersants are used. In practice, the limit of simple gravity methods is 
fractionation up to approximately 1 |Lim. Under certain conditions, ultra- 
centrifugation makes it possible to reach the nanometric domain. 

Fractions below 0.5 |Lim contain practically no more quartz or primary 
minerals. Fractions below 0.2 |Lim enable better characterization of 
argillization horizons. This threshold, proposed around 1931, was at that 
time regarded as representative of the limit of "the colloidal state" because 
after elimination of oxides and hydroxides, the fraction presented 
homogeneous chemical composition comparable to a mono-dispersed 
system, i.e. the same exchange capacity, the same structural composition of 
the 0.2 |Lim, 0.1 |Lim, and 0.05 |Lim particles. 

When the final purpose of fractionation of the particles is physical or 
chemical determination, the different treatments that are carried out to put 



66 Mineralogical Analysis 

the very fine fractions in suspension can differ considerably from the 
methods used for textural analysis because secondary products cannot be 
significantly modified, in particular clays and oxides. In certain cases, it is 
possible to simply put fine fractions in suspension by ultrasound and to use 
ion exchange resin for desaturation. Dispersants of the hexametaphosphate 
or pyrophosphate type (cf Chap. 2) should not be used. 

The criteria of Stokes law are suitable for centrifugation and the same 
problems will occur with particles whose speed is changing. By quantitatively 
isolating all the fractions, chemical and physical determination can be 
refined, and more detailed distribution curves established for the particle 
continuum. 



3.2 Fractionation by Continuous Centrifugation 



3.2.1 Principle 

After the treatments needed to isolate the primary particles (cf. Chap. 2), 
the sample is put in suspension for later analyses using a non- 
contaminating dispersant. The fine fractions (less than 2 |Lim) are first 
separated from the coarse fractions by several siphoning operations, which 
take the falling time of the silts into account. Five to six siphonings are 
sufficient for a quantitative recovery. 

Because of the volumes being dealt with, fractionation of the fine phases 
is carried out in a centrifuge with continuous inputs per ascensum made up 
of a vertical tube able to rotate at 52,000g (Fig. 3.1a). A transfer paper 
placed inside the tube makes it possible to collect the particles that 
sediment on the wall (Fig. 3.1b). The effluents are collected at the top of 
the bowl by a single or double chute (depending on the model). 

The suspension does not completely fill the tube but forms a concentric 
ring, the centre being a cylinder of air, Ri (Fig. 3.1b). The thickness of the 
film of suspension is R 2 - R\. The particles gradually sediment on the walls 
of the bowl as a function of their density and of their diameter. They follow 
a parabolic trajectory starting from the point of deposit of the effluents. 

The radial application of the centrifugal force is accompanied by a 
vertical force. A particle of a given diameter and density will deposit 
according to the resulting force vector. Viscosity can be modified in the 
course of centrifugation by variations in temperature, the influence of the 



Fractionation of the Colloidal Systems 



67 



pressures created at high speeds and possibly by the presence of thixotropic 
materials. With soils containing allophanes, it is sometimes possible to 
observe flocculation if the dispersing medium is not homogeneous 
throughout the iterative extractions, or due to loss of part of the swelling 
water. It should be noted that fibres of imogolite, mica plates, tests of 
diatoms, etc. do not follow Stokes' law and are separated in a random way. 




Fig. 3.1. (a) Uninterrupted ultracentrifuge system using compressed air - 45,000g 
(1 input nozzle for injection; 2, 3 exit chutes for effluents; 4 rev counter; 5 
connecting with bowl; 6 centrifugation bowl), (b) Centrifugation bowl with 
transfer paper for recovery (division of the delivery points according to 
procedure in "Fractionation in Four Phases"). 



The component perpendicular to the axis can be easily calculated 
starting from Stokes law (cf. Sect. 3.2.2), however, it should be noted that 
with centrifugation, the deposit rate of the particles is not constant due to 
variations in the intensity of the field of centrifugation which depend on 
the diameter of the rotor. The vertical component must be calculated as a 
function of the flow, which itself depends on the input, on the diameter of 
the injector channel, and on the diameter of the ring outfall. 



68 Mineralogical Analysis 

In practice, the formulas for computation of the standard Sharpies 
centrifuge cannot be rigorously applied at the bottom and the top of the 
bowl because of turbulence at these levels (injector channel and deflector at 
the bottom, ring outfall at the top). Too much deposit can also modify the 
radius at the bottom of the bowl. However, the height of the deposit can be 
determined with sufficient accuracy by following a strict procedure. It may 
be advantageous to collect a relatively small quantity of sediments (not 
exceeding 10 g) at each treatment. 

Under certain operating conditions (number of revolutions, flow, etc.), 
the finest particles can pass across the bowl without sedimentation because 
the time of passage is insufficient for the centrifugal force to transfer them 
to the wall. However, if one of the variables is modified, for example, the 
flow is slowed down by changing the input tube; these particles can also be 
collected. With a low flow, separation is differentiated more satisfactorily 
than with a high flow. For satisfactory separation, it is better to use tubes 
with a small diameter and a not too high charge. For simple separation (for 
purification by enrichment) a 2 mm tube and a higher charge (75 cm for 
example) can be used. The drain may still contain fine particles depending 
on the number of revolutions applied. The system makes it possible to use 
the drain again with a lower flow rate and to collect the solid fraction on a 
new transfer paper placed in the bowl. 

Table 3.1. Deposit of particles of various minerals by continuous ultracentrifugation 
(flow 730 ml_ min -1 speed 20,000g, viscosity 1) 

mineral density diameter of particles which sediment at a 

height of 10 cm in the bowl (in jim) 



opal 


2.10 


360 


gibbsite 


2.40 


320 


quartz 


2.65 (average used 
for the soils) 


280 


hematite 


5.26 


115 



It is important to choose a speed and a flow which allow non-uniform 
deposit to obtain satisfactory classification of the particles. It is difficult 
to include all the variables in the calculation, and the forecast will be 
uncertain if the average density of 2.65 used for the soils is not regular. 
Indeed, if a mixture of minerals of different densities is used whose 



Fractionation of the Colloidal Systems 69 

micro-particles are individualized in the suspension, very different particles 
with the same diameter will be found at any level (Table 3.1). 

At one time, it seemed that graphic methods would be both adequate and 
fast. Saunders (1948) studied a nomographical representation applicable to 
continuous ultracentrifugation (1936-1940), and was able to determine that 
certain factors in the equation of Hauser and Lynn (cf. Sect. 3.2.2) 
remained constant for a given procedure thus allowing simplification and a 
move to a nomographical representation with five variables that could be 
extended to six variables using the method of Davis (1943). 

It thus became possible to rapidly determine the height of deposit of 
elementary particles with different densities but the same diameter. 



3.2.2 Theory 

The method of Hauser and Lynn (1940) to calculate the size of the particles 
is one of the most powerful for use with an ultracentrifuge with continuous 
input. The equation makes it possible to express the vertical distance (7 in 
cm) from the deposit of a particle of a given size, measured starting from 
the bottom of the bowl of the centrifugal machine by 



18 7^7/ 
k{R\ -Rl)D 2 co 2 Sp 



Y =C — J* # 2o (3.1) 



where 



7v~ 7v~ R, 7v~ X n — R n 

C=^ln^-^ln^- +^ 2 - (3.1') 

2 X 2{ Xj 4 

R 2 = distance from the axis of rotation to the side of the bowl (cm); 

Ri = distance from the axis of rotation on the surface of the liquid in the 
bowl (cm); 

X = distance from the axis of rotation at which a given particle must 
start to deposit on the side of the bowl (cm); 

K x = function of the construction of the bowl (cm 2 ) 



R 2 -R x 



(3.2) 



(3/4)7?; + (l/4)7? 2 4 -R?R 2 2 -R? ln(7? 1 1 R 2 ) 
Q = throughput speed (flow of the suspension mL s 1 ); 
?] = viscosity of the dispersion medium (poise); 

D = sphere equivalent diameter of the particles which sediment at 7 cm; 
co = angular speed of rotation (radian per second); 



70 



Mineralogical Analysis 



Sp = difference in density between the dispersed particles (Table 3.2) 
and the medium of dispersion (g mL" 1 ). 

Under standard conditions (flow and range of the particles), the equation 
of Hauser and Lynn is related to X and D. For particles of a given 
diameter, the equation is a function of only X , which makes it possible to 
plot the curve of C vs Y. On this basis, Saunders (1948) established a 
system of monograms which allows the equivalent diameter of the particles 
settling in the bowl to be calculated with satisfactory precision. 

io F 

By defining the constant A = ^—^ — 1 —^\> 0.1) becomes: 



n(R 2 2 -R?) 



Y = A T] Q 
C D 2 co 2 Sp 



(3.3) 



Table 3.2. Specific density of some minerals (average density used for the 
soils = 2.65) 



minerals 


density 


minerals 


density 


Al boehmite ALO(OH) 


3.07 Fe 


akaganeite 
Fe 3+ 0(OH, CI) 


3.55 


diaspore AIO(OH) 


3.40 


goethite FeO(OH) 


4.0- 
4.4 


bayerite AI(OH) 3 


2.53 


lepidocrocite FeO(OH) 


3.85 


gibbsite AI(OH) 3 


2.40 


hematite Fe 2 3 


5.26 


nordstrandite AI(OH) 3 


2.43 


maghemite Fe 2 3 


5.10 


corundum Al 2 3 


4.10 


magnetite Fe 3 4 
(Fe 2+ Fe 2 3+ 4 ) 


5.17 


akdalaite 
4AI 2 3) H 2 


3.68 


ilmenite FeO-Ti0 2 


4.44- 
4.90 


bauxite Al 2 3) 2H 2 


2.55 


pyrite FeS 2 


5.02 


Mn manganosite MnO 


5.36 clays kaolinite 


2.60 


pyrolusite Mn0 2 


5.06 


halloysite 


2.10 


ramsdelite Mn0 2 


4.50 


dickite 


2.60 


manganite MnO(OH) 


4.33 


montmorillonite 


2-3.00 


feitknechtite 
MnO(OH) 


3.80 


nontronite 


2-3.00 


groutite MnO(OH) 


4.15 


beidellite 


2-3.00 



Fractionation of the Colloidal Systems 



71 



pyrochlorite Mn(OH) 2 


3.25 


micas: 


biotite 


3.00 


nsutite 
Mn +2 Mn +4 2 (OH) 2 


4.50 




phlogopite 


2.80 


hausmanite 
Mn +2 Mn 2 +3 4 


4.84 




glauconite 


2.60 









muscovite 


2.80 








illite 


2.6- 
2.9 


Si 


coesite Si0 2 


2.93 


feldspar 
white feldspar 
Na 2 0, Al 2 3) 6Si0 2 


2.61- 
2.64 




cristobalite Si0 2 


2.33 


andesine (CaO, Na 2 
Al 2 3) 4Si0 2 


2.65- 
2.69 




quartz Si0 2 


2.65 


oligoclase NaAISi 3 8 + 
CaAI 2 Si 2 8 


2.62- 
2.67 




tridymite Si0 2 


2.26 


orthoclase KAISi 3 8 


2.56 




silhydrite 3Si0 2 , H 2 


2.14 








opal Si0 2) nH 2 


2.10 






Ca 


calcite CaC0 3 
aragonite CaC0 3 


2.7-2.9 






2.9 


gypsum CaS0 4 , 2H 2 


2.3 



This equation can be treated using the "nomographical method" 
according to Davis (1943) and gives a value of YIC to each value of Y. If 7] 
is expressed in centipoises, Q in mL min _1 , D in nm, co in rotations min 1 , 
Sp in g mL" 1 Y in cm and C in cm 2 the equation is written: 



Y -= 1.52X10' 2 A 2 \ Q 
C D 2 a) 2 8p 



(3.3') 



This equation can be reduced to four equations with three variables and 
three parameters a, (3 and y : 

log a = log ?] - log% 

log jS = log Sp -2 log co 

log y - log a + log j3 

2\ogD = log y + log Q 

The nomographical method of Davis (1943) provides a solution by 
tracing four lines which represent the solution of one of these equations 
(Fig. 3.2). Constant A is included starting from a scale representing a 
numerical solution of (3.3'). For example, for a centrifugation tube where 
R { =2.175 cm and R 2 = 0.735 cm, constant A is equal to 2.44 x 10 12 . The 
final formula for direct calculation will be: 



72 



Mineralogical Analysis 



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Fractionation of the Colloidal Systems 



73 



D = 2.44x10' 



TlQ 



{co 2 s P y c f 



with value Y/C for the height Y and the units of (3.3'). 

3.2.3 Equipment and reagents 

Equipment 

-Standard Sharpies Tl ultracentrifuge with continuous circulation and a 
turbine equipped with an 8RY stainless steel bowl with a diameter of 44 
mm 

- 6 L Pyrex bottle with broad neck with stopper 

- Stopper pierced with a glass tube with an interior diameter of 10 mm cut 
in bevel (for input of suspension) 

- 12 cm Conical forceps with jaw punts (to retrieve the transfer paper) 

- Small plastic spatula 

- Stylet (to lift the transfer paper for removal with the grip) 



J 


t ' ' ' 
1 j J 1 

1 1 1 1 

: V 

i 

r 
i 


9 i « * 1 

- 

I 

■i 



Fig. 3.3. Spectra of X-ray diffraction: 

dashed line = Tymek support alone, 

^^^^ solid line = kaolinite on Tymek support (sufficiently thick to avoid 
contamination). 



74 



Mineralogical Analysis 



- Plastic transfer papers with frosted interior (Integraph, Invar 75 |Lim, 
Kodatrace, Chronaflex or Tymek Dupont de Nemours) 204x150 mm 
sheets; the thickness of this support must be homogeneous, it must have a 
constant weight per surface unit, be resistant to water, have a flat DRX 
spectrum or well-defined peaks outside the zones of measurement of the 
sample (Fig. 3.3) 

- 1/10 mg balances 

- Set of suitable pillboxes and micro-bottles (plastic, single use, Fig. 3.4) 



Preparation 
grids TtM-STEM 

Powder XRD 
Palletizing IR 
SEM + 
mlcroprobe 
Oriented 
XRD 

All analysis 
on clay 
powder or 
clay flak© 







3 





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A 





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Dry Wet 



24x24 
mm 




Al support 
SEM preparations 
transfer paper 1 
transfer paper 2-3 
transfer paper 4 



Carbon lake 
sticking 



Recovery of 

residues 

on zones A, B, C 

of transfer paper 



Fig. 3.4. Example of system for processing the fine fractions separated on transfer 
paper (Fig. 3.1), 1 = regrouping on MEB support, 2 = only andisols- 
histosols, 3 = pillbox, micro-bottles 

- Aluminium supports for scanning electron microscopy 



Reagents 

Cf. Chap. 2 



Fractionation of the Colloidal Systems 75 

3.2.4 Procedure 



Standard Continuous Ultracenthfugation 

- Choose the diameter of the injector channel, the diameter of the ring 
outfall, and the height h of level X (Fig. 3.1). 

-Place in the bowl of the centrifuge a tared transfer paper with the selected 
cutting plan drawn on the back (the weight of the transfer paper P 
makes it possible to calculate the weights P ou P 02 , P03, corresponding to 
the respective surfaces of zones A, B, C in Fig. 3.1). 

- Suspend the bowl on the rotor and place the container for the recovery 
of the effluents under the chute. 

- Fill the funnel to a constant level X with the same dispersant as the 
samples, taking care not to trap air in the adduction tube. 

- Homogenize the bottle containing the clay suspension. 

- Remove an aliquot of 2 mL (for grid TEM). 

- Rock the bottle on the funnel; switch on the centrifugal machine at the 
selected speed. 

- Place the injector channel under the bowl and open the input cock. 

When all the liquid has gone, add 200 mL of the dispersion liquid to 
drive out all the suspension remaining in the bowl (approximately 150 mL). 
Adjust to a maximum speed of 52,000g for 2-3 min to stabilize the deposit. 

Disconnect the input tube and stop the centrifuge (collect the liquid 
remaining in the tube in a crystallizer and discard it if it is clear). 

Remove the transfer paper carefully by holding the bowl obliquely to 
not contaminate the top of the bowl with coarse particles. Spread the paper 
out flat. Recover any trace of deposit on the deflector and add it to the 
bottom of the transfer paper. 

Leave to dry at room temperature (if necessary recover wet clay with a 
spatula and place it on the appropriate zone of the transfer paper before 
drying). 

Weigh the transfer paper and dried clay: P\. Deposited clay corresponds 

toPi-Po 

Cut the transfer paper following the plan on the back. This makes it 
possible to weigh zones A, B, C (Fig. 3.1), i.e. P n , P\i, P13. 

Continuous Fractionation of the Colloidal Particles 



The complex and time-consuming method of Hauser and Lynn (1940) 
enables isolation of the fine fractions from a suspension (Fig. 3.5) and the 



76 Mineralogical Analysis 

establishment of cumulative curves of distribution of the particles which 
can nowadays be accomplished continuously with an automatic apparatus 
for the measurement of particle size (cf. Chap. 2). This type of apparatus 
has two centrifugation speeds and seven different flows, i.e. 1 1 passages in 
the centrifuge to classify particles between 1 |Lim and 24 nm with a 
Sharpies continuous centrifuge with a turbine. 

This method cannot be used for routine tasks because of the length of the 
operations, or for fragile samples that are difficult to maintain in 
suspension like soils with allophane. 

The method is nevertheless useful in metallogeny to identify the 
enriched fractions (release mesh). The suspensions must be diluted to 
accomplish fractionation without an awkward piston effect. 

Fractionation in Four Phases 

The method of Gautheyrou and Gautheyrou (1967) is based on the 
equations of Hauser and Lynn and the nomographical system of Saunders 
(cf. Sect. 3.2.2). The transfer paper shown in Fig. 3.1b is an example of one 
layout suited to the needs of mineralogical analysis, but other alternatives 
are possible considering that the particles deposited in a horizontal plane 
are similar and that the solid phase varies upwards. 

Zones A, B, C (Figs. 3.1 and 3.4) allow separation of the particle size 
phases whose significance depends on the procedure used (these fractions 
are used for chemical and physical analyses). Zones A' and C (Figs. 3.1 
and 3.4) enable fractions to be isolated for X-ray diffraction either after 
crushing for powder diagrams, or pretreatments, or directly for oriented 
diagrams on the 24 x 24 mm 2 (cf. Chap. 4). After sticking the 5 x 5 mm 2 
on a suitable support with carbon lacquer (cf. Chap. 8) they can be used for 
electronic scan microscopy with EDX microanalysis. 

After dilution and preparation of the grids, a 1 mL sample of each 
suspension enables observation by transmission electronic microscopy (cf. 
Chap. 8). 

Adjustments 

Charge h: 75 cm (Fig. 3.1); temperature: 20°C; tube: 2 mm diameter; 
flow: 730 mL min" 1 ; time of passage in the bowl: 15 s; average density: 
2.65; ring outfall: 0. 

Charge, flow, temperature remain constant; only the speed is modified at 
each centrifugation: 6,000, 10,000, 25,000, 50,000g (at 50,000g, if the 
sample contains very fine particles, it may be necessary to use a 1 mm tube 
corresponding to a flow of 160 mL per minute to fix all the very fine 
phase). 



Fractionation of the Colloidal Systems 



77 











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a 

a: 




V 3, 






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43 nm 116 nm 95 nm 75 nm 58 nm 47 nm 34 nm < 24 
16 nm 95 nm 75 nm 58 nm 47 nm 34 nm 24 nm 


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t 

S 


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7g 














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100 cm 3 min" 1 
















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200 cm 3 min 1 








LITa 












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8^ 1 


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13 


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suspension 




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Diagram of fractionation 
by method "Continuous 
Fractionation of the 
Colloidal Particles" 




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78 Mineralogical Analysis 

Fraction 2-1 |im 

This fraction is separated at a centrifugation speed 6,000g and recovered on 
the first transfer paper (TP): weight TP alone: P ou TP + deposit: P n , 
weight clay: 2-1 |Lim: Pn-Poi • Pci Express in % compared to the initial 
weight of soil. 

Fraction 1-0.5 \im 

Recovered on the second TP at 10,000g: Pc 2 

Fraction 0.5-0.2 |im 

On the third TP at 25,000g: Pc 3 

Fraction 0.2-0.05 |im 

On the fourth TP at 50,000g and lower flow of 160 mL min 1 , 1 mm tube: 
Pc 4 

Clay < 0.05 \im 

This very fine clay is contained in the last draining water and can be 
recovered by flocculation. This fraction is generally a clear extract that can 
be discarded because it contains the majority of the residual impurities of 
the reagents used for the initial preparation of clay. Electronic microscopy 
can also be used, but concentration by evaporation can result in risks of 
hydrolysis and neo-formation. 

Remarks 

If the deposit is too thick, irregular cracking can occur in clays with a high 
shrinkage coefficient, which makes it impossible to cut the 5x5 mm 2 for 
oriented XRD. In this case, the squares are not used but instead a sliver of 
clay is stuck down with carbon lacquer. 

In certain soil samples the finest fractions are practically non-existent. In 
this case it is possible to stop at the second or the third centrifugation. 

If major variations are observed between the coarsest phase and the 
finest phase, preparation and analyses of sub-samples should be performed 
progressively. 

Certain studies may require conservation of part of fresh clay without 
drying. In this case, the transfer paper should have an additional vertical 
separation. Fresh clay should be recovered immediately at the outlet of the 
bowl with a plastic spatula and quickly stored in a pillbox. Quantification 
can be checked on the fraction which is dried but there is a risk of error due 
to transformations during drying. 



Fractionation of the Colloidal Systems 79 

The same method can also be used to: 

- Separate and enrich two phases; for example two phases with the same 
mineral density but different particle size (micro-micas and coarser 
crystallized kaolinite). 

- Separate two products of the same particle size but different density, for 
example aluminous products with a density <3.0 and ferrous or ferric 
products with a density of 4-5. 

- Collect the particles of a given diameter and density on a narrow zone 
(metallogeny - separation meshes), etc. 

Chlorite, vermiculite, or kaolinite enrichment is often observed in the 
coarse fractions. Well-crystallized kaolinite is generally present in fractions 
>0.5 |Lim. Quartz and muscovite are almost eliminated in the fractions <0.5 
|Lim, which makes it possible "to clean" the spectra. 

Gibbsite is mostly retained in the coarse fractions and is only present in 
very small amounts in the very fine phases. 

Halloysite enrichment can be also observed, as well as enrichments in 
substances with short distance crystalline arrangement in fractions <0.5 |Lim 
or 0.2 |Lim, whereas well-crystallized kaolinite disappears. 

Deferrization increases the smoothness of the particles revealing the 
incorporating effect of iron. 

Studying the different fractions by X-ray diffraction enables observation 
of possible crystallochemical heterogeneity of the colloidal fraction and 
identification of mineral filiations which occurred during evolution and 
weathering. Minerals whose spectral signature is readable only above 5% 
(for example Sepiolite) can also be detected. 

Fractionation with Different Flows 

In this technique (Biological Centre of Pedology, Nancy, France), the clay 
suspension >2 |Lim at a concentration not exceeding 1%, is subjected to an 
initial series of centrifugation at fixed speed and flows (Fig. 3.6). 

A second series of centrifugation with a much lower flow (3 1 mL min" 1 ) 
makes it possible to separate the finer fractions. Thanks to repeated 
centrifugation and the weak concentration of the medium, the separation of 
the fractions is considered quantitative. 



3.3. Pretreatment of the extracted phases 

Mineralogical analyses performed directly on total soil samples provide 
information on all the most abundant components, but do not allow 



80 Mineralogical Analysis 

detection of the presence of low concentration phases because of the lack 
of sensitivity of the instrumental techniques and the occurrence of much 
interference. These low-concentration phases (<5 %), which can be highly 
significant in explaining certain processes of soil genesis, are masked by 
background noise even when they are above their threshold of detection. 

It is thus necessary to eliminate interference and to concentrate the 
"mineralogical clay" phase. 

Using a suitable fractionation method, clays are purified and 
concentrated and appear in a homoionic form: NH 4 + , Ca 2+ , or H + depending 
on the case. As mineral cements and the organomineral links have been 
destroyed, it is possible to obtain satisfactory separation of the particles. 

The use of ultrasound enables good separation of elementary particles, 
and complementary pretreatments can be performed on clays to allow the 
use of specific instrumental techniques. 

Selective dissolution makes it possible to eliminate the iron oxides 
which can obstruct XRD (cf. Chap. 4), fluorescence with a copper tube) 
and DTA-TGA (cf. Chap. 7), oxidation Fe 2+ ). Different pretreatments 
make it possible to carry out analyses using NMR, ESR, Mossbauer, etc. 

Gels and substances that are amorphous to X-ray and have crystal 
lattices with short distance arrangement can be dissolved, enabling their 
quantification and the production of differential spectra (DXRD) to 
identify them. 

Any calcium carbonate that is still present can be eliminated by 
complexing Ca 2+ ions with a solution of normal EDTA. 

Pretreatments also help improve the orientation of clays (which is 
disturbed by iron, for example in coatings), the intensity of the spectra of 
diffraction and the ratio of diffraction to background noise. 

Clays often have to be studied after homoionic saturation by a cation 
(such as Mg 2+ which regulates the adsorption of water by clays with an 
expansible interfoliaceous space, or K + which limits the adsorption of 
water and thus the swelling of the layers). 

Other treatments, like solvation by polar solvents, or the creation of 
complexes of intercalation, make it possible to identify certain clays. Heat 
treatments are also used specifically to cause the collapse of the lattices or 
to modify surface properties. These methods are described in detail in 
Chaps. 4-7. 



Fractionation of the Colloidal Systems 81 

References 

Davis DS (1943) Empirical Equations and Nomography. Mc Graw Hill, 

New York, 1, 104-114 
Gautheyrou J and Gautheyrou M (1967) Mode operatoire pour V extraction et la 

purification de la fraction argileuse < 2 jum. Notes de laboratoire, 

Orstom-Guadeloupe, mars 1968, 1-9, Orstom 
Hauser EA and Lynn JE (1940) Separation and fractionation of colloidal 

systems. Ind. Eng. Chem., 32, 659-662 
Saunders E (1948) Nomograph for particle size determination with the Sharpies 

supercentrifuge. Anal Chem., 20, 379-381 



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82 Mineralogical Analysis 

Johnson L (1956) Particle size analysis and centrifugal sedimentation. Trans. Bull. 

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Muog E, Taylor JR, Pearson RW, Weeks AE and Simonson RW (1936) Procedure 

for special type of mechanical and mineralogical soil analysis. Soil Sci. 

Soc. Am. Proc, 101-112 
Rouiller J, Brethes A, Burtin G and Guillet B (1984) Fractionnement des argiles 

par ultra-centrifugation en continu : evolution des illites en milieu 

podzolique. Sci. Geol. Bull, 37, 319-331 
Schachman HK (1948) Determination of sedimentation constants in the Sharpies 

supercentrifuge. J. Phys. Colloid. Chem., 52, 1034-1045 
Tan KH (1996) Soil Sampling, Preparation and Analysis. Dekker, New York, 

278-361 
Tanner CB and Jackson ML (1947) Nomographs of sedimentation times for soil 

particles under gravity or centrifugal acceleration. Soil Sci. Soc. Am. 

Proc, 12, 60-65 
Tran-Vinh-Ann and Ndejuru E (1972) Analyse granulometrique de la fraction 

argileuse par centrifugation en flux continu. Mise au point d'une methode 

et application a quelques sols tropicaux. Pedologie, XXII, 366-382 
Truo E, Taylor JR, Simonson RW and Week ME (1936) Mechanical and 

mineralogical subdivision of the clay separate of soils. Soil Sci. Soc. 

Proc, 175-179 
Tu Y, O'Carroll JB, Kotlyar LS, Sparks BD, Ng S, Chung KH and Cuddy G 

(2005) Recovery of bitumen from oilsands: gelation of ultra- fine clay in 

the primary separation vessel. Fuel, 84, 653-660 
Whittig LD and Allardice WR (1986) X-Ray diffraction techniques - separation 

of particle - size fraction. In Klute A (ed.), Method of Soil Analysis Part 

Physical and Mineralogical Methods, second edition, American Society 

or Agronomy, 340-342 



Mineralogical Characterization by X-Ray 
Diffractometry 



4.1 Introduction 



4.1.1 X-Ray Diffraction and Mineralogy 

Methods using optical microscopy in petrography are not suitable for the 
identification of mineralogical clays with small particles whose crystal 
lattices vary with water content and with their ionic environment and 
whose chemical composition is often unclear (Tables 4.1 and 4.2). 
Among other available methods, X-ray diffraction (XRD) is one of the 
most efficient. Coherent scattering of the incidental radiation in XRD 
makes it possible to clearly identify both the parameters of the crystal 
lattice and the geometrical distribution of the atoms in the crystal mesh. 

XRD can be combined with or supplemented by geochemical and 
isotopic analyses (AAS, ICP, ICP-MS, EXAFS, etc.), thermal analyses 
(DTA-TGA, DSC, EGD, EGA, etc.), analyses that enable evaluation of 
interatomic or intermolecular binding energies and order-disorder relations 
(e.g. FTIR, Raman spectrometry, Mossbauer spectrometry, NMR), high 
resolution transmission electronic microscopy (+ electron micro- 
diffraction) and electronic scan microscopy. EDX or WDX probes make 
it possible to link in situ chemical composition with the shapes of the 
particles to be observed. Total chemical composition is determined by 
total analyses after mineralization in mediums that enable solubilization 
of all the components; selective dissolution makes it possible to sub- 
divide the sample into fractions of different chemical resistance; these 
sub-divisions are essential both for quantification and for purification of 
the samples before analysis by instrumental methods (e.g. XRD, IR or 
NMR spectroscopy). 



84 



Mineralogical Analysis 



Table 4.1. Classification of clays proposed by the international association for 
the study of clays (AIPEA 1 ) 



type 



group (x = charge 
by unit formula) 



sub-group (n = number of 
cations of octahedral layers) 



species 



1:1 



kaolinite - 
serpentine 
(x=0) 



kaolinite (n = 2) 
serpentine (n = 3) 



kaolinite, halloysite, 
chrysotile, lizardite, 
antigorite 



2:1 



pyrophyllite - Talc 

(x=0) 

smectites 

montmorillonite 

saponite 

(x = 0.25-0.6) 

vermiculite 

(x = 0.6-0.9) 



micas 2 (x= 1) 



pyrophillite (n = 2) 

talc (n = 3) 

dioctahedral smectites or 

montmorillonites (n = 3) 

trioctahedral smectites or 

saponite (n = 3) 

dioctahedral vermiculites (n 

2) 

trioctahedral vermiculites (n 

3) 

dioctahedral micas (n = 2) 



pyrophillite 

talc 

montmorillonite, 

beidellite, nontronite 

saponite, hectorite, 

sauconite 

dioctahedral 

vermiculite 

trioctahedral 

vermiculite 

muscovite, 
paragonite 



breakable micas 
(x = 2) 



trioctahedral micas (n = 3) 
dioctahedral breakable micas 

(A7 = 2) 

trioctahedral breakable micas clintonite 

(A7 = 3) 



biotite, phlogopite 
margarite 



2:1:1 



chlorite (x variable) 



dioctahedral chlorites (4<n<5) 
di-trioctahedral chlorites 
trioctahedral chlorites (5 < n < 6) 



donbassite 

cookeite, sudoite 

penninite, 

clinochlore, 

prochlorite 



Inter-analytical tests can be performed at different levels: on the 
structure of clays (geochemical relations, pedological differentiation 



1 AIPEA Association Internationale pour VEtude des Argiles, GPO Box 2434, 
Brisbane, Qld4001 Australia 

2 Illites (or hydromica), Sericite, etc., many materials labelled illites can be inter- 
stratified. 



X-ray Diffractometry 



85 



within a profile and spatial differentiation), hydrous properties (porosity, 
permeability, functional waterlogging generated by the nature and the 
proportion of clays), adsorbing complexes (charge distribution, CEC, 
etc.). 

For a detailed quantitative study, in addition to the clay particle 
fraction, it is generally necessary to analyse the fine silt fraction which 
can contain interstratified minerals, particularly in the case of micas and 
well crystallized kaolinite. A balance takes into account clay and 
associated phases, oxides, hydroxides, etc. making it possible to explain 
apparently unmatched results (excessive Si 4+ content due to diatoms, very 
fine quartz, high percentages of K + originating from potassic feldspars, 
micas, etc.). 

Table. 4.2. Structural lexicon (AIPEA, 1972) 



English 


French 


(atomic) plane 


plan (atomique) 


(tetrahedral or octahedral) sheet 
(plane combination) 


couche (tetraedrique ou 
octaedrique.combinaison de plans) 


1:1 or 2:1 layer (sheet combination) 


feuillet 1:1 ou 2:1 (combinaison de 
couches) 


interlayer space 


espace interfoliaire 


unit structure = combination of layers 
+ interlayer materials 


assemblage de feuillets + materiel inter- 
foliaire = unite Structural 


lattice 


Reseau 



Fig. 4.1. The crystal mesh 




86 Mineralogical Analysis 

4.1.2 Principle 

A crystal is defined as a solid made up of atoms assembled in a three- 
dimensional periodic model. Lengths a, b, c and angles a, /?, ^between 
the planes define the mesh parameters of the basic unit (Fig. 4.1). 

When monochromatic X-ray beams of suitable wavelength strike a 
crystalline plane, the X-rays are reflected by the atoms of the crystal. The 
signal is reinforced in a particular direction if the rays reflected by the 
different planes (Fig. 4.2) are in phase. This phenomenon corresponds to 
Bragg 's law 

2 dsinz? = nA (4.1) 

where d is the space between atomic planes or the inter-reticular distance 
in the crystal (d(hkl)); X is wavelength and 6 is angle between beam and 
atomic plane and n is the order of diffraction (integer number). 

All the planes of a crystal diffract the X-ray when the crystal is tilted at 
certain angles of the incidental beam of wavelength A in accordance 
with the law (4.1). 

The angles 6 are linked to wavelength X and distance d, which are 
expressed in Angstroms or nanometres (1 A = 0.1 nm = 10" 10 m). If the 
wavelength is known, measuring the angle of reflection makes it possible 
to determine the inter-reticular spaces. 

Remarks 

Certain minerals can be "amorphous" to X-ray either because they do not 
have a specific crystalline arrangement (true of glasses) or because they 
have short-range organization that is too small to be detected at a 
wavelength of 1-2 A. 

XRD is not the best technique for the study of non-crystalline solids 
such as allophanes which are made up of clusters of Si atoms presenting 
structural elements with interlayer distances corresponding to 1 or 2 
neighbouring atoms. 

Atoms of silicon in a tetrahedral position and atoms of aluminium in 
octahedral coordination but with no regular symmetry, cannot give well 
defined peaks, but only broad and badly defined peaks that appear around 
0.33 and 0.22 nm. 



X-ray Diffractometry 



87 



4.1.3 XRD Instrumentation 



X-rays were discovered in 1895 by Roentgen, but the phenomenon of 
crystal diffraction was discovered only in 1912. 



Incident 
beam 



X-ray 




Diffracted 

beam 



Fig. 4.2 Diffraction of an incidental beam by crystalline reticular planes. Lines p, 
p1, p2 represent the parallel and equidistant reticular planes separated 
by space d. An X-ray beam striking the higher plane p will be reflected in 
the incidental angle 6. To obtain a measurable reflection, all the rays 
reflected by planes p, p1, p2, etc, must be in phase. To achieve this, 
GE + EH, the path difference between radiations ABC and DEF must be 
equal to a whole number of the wavelength. As GE = EH = d sin 6, the 
condition is thus the law of Bragg (1 ). 



X-rays are located between UV and gamma radiation wavelengths, 
i.e. approximately 10 to 10" 2 nm. In XRD, "hard" radiation is used with 
wavelengths from 1.5 to 1.9 A depending on the anti-cathode used (1 A = 
0.1 nm). Radiation X-ray is propagated in a straight line. It is necessary to 
ensure the radiation is as monochromatic as possible by using a set of 
filters, slits and a monochromator. The apparatus comprises: 

- a generator with stabilized high voltage and micro-intensity which 
supplies the X-ray tube and the counter 

- a sealed X-ray tube, including a source of electrons maintained from 20 
to 50 kV by means of a high negative potential and an anode or anti- 
cathode (Table 4.3) made of thick metal, cooled by a water circuit; 



Mineralogical Analysis 



high speed bombardment of the electrons causes transfer of energy to 
the atoms of the anode-target bringing them to a higher energy level, 
thus creating orbital vacancies of electrons; the quantum of energy 
produced is characteristic of the atoms of the anode; X-ray photons 
leave the tube by 300 |um thick beryllium windows that are transparent 
to the X-ray (the spot must be as small as possible to concentrate the 
energy of the electrons on a limited zone of the anode and to ensure a 
high intensity X-ray source); the power of the tube is limited by the 
quantity of heat likely to be dissipated by the anode and is expressed by 
the maximum acceptable value in mA for a given voltage (1-3 kV or 
more in the case of a rotating anode); characteristic radiations are 
obtained only starting from a given critical voltage of excitation, thus 
strict regulation of the voltage and intensity is required to avoid 
modifying the wavelength 

- a goniometer, which makes it possible to rotate the sample and the 
counter under the conditions fixed by the Bragg equation; this provides 
a support both for the sample whose plane is adjusted very precisely 
and for the focused detector which turns around the same axis in the 
same direction at a suitable speed ratio 

- a linear beam is obtained by means of the Soller slits and the degree of 
divergence which limits the opening of the beam; variable slits are now 
used with openings linked to the angle making it possible to irradiate a 
constant surface, which is particularly useful with small angles because 
the effects of the direct beam are limited 

- a reception slit limits the width of the beam in the focal plane; the 
narrower the slit, the higher the resolution, though there is a loss in 
intensity; a graphite back monochromator limits fluorescence radiation, 
incoherent radiations and the Compton effect; the perfect alignment of 
all the different elements determines the quality of the measurements 

- a detection system (counter) makes it possible to measure the intensity 
of the X-ray transmitted; the number of pulsations per unit of time is 
proportional to the quantity of X-ray transmitted; the counter can be 
linear or proportional, or detection can be by scintillation, or 
occasionally by semiconductor (semiconductor counter must be kept in 
liquid nitrogen at -196°C). 

Safety 

X-ray radiation is dangerous and can cause burns, genetic modifications, 
cancers, etc. The risks associated with high voltage must be also taken 
into account. 

Careful prevention is essential and strict regulations apply to all 
apparatuses, which must be equipped with safety devices: 



X-ray Diffractometry 



the sealed tube must be protected by thick walls to eliminate risk of 

radiation 

the goniometer must be insulated with lead glass or lead plastic to 

protect the whole apparatus; it should only be opened when the tube is 

switched off 

the operator must wear a monitoring badge with a sensitive film 

(dosimeter) that accounts for possible whole-body irradiation (which 

must be checked regularly), as well as a ring to measure irradiation of 

the hands 

operators must have regular medical check-ups to detect changes in the 

blood count (white blood cell count, etc.) 

a Geiger counter must be used to check for radiation leaks: fluorescent 

screens should be placed on supports made of zinc doped with nickel to 

identify the zones struck by the beam while alignment is being 

adjusted. 

Table 4.3. Characteristics of some anti-cathodes 



anti- Ka K induced excitation operating 

.. . wavelength P ,, potential potential remarks 

cathode 2 a filter fluorescence „ XXM v^ wx 

A (KV) (KV) 

penetration 
and average 
Cu 1.542 Ni Co-Fe 9.0 25-45 dispersion, 

not much 
affected by air 



Co 



1.791 



Fe 



Mn-Cr 



7.8 



25-35 



weak 

penetration 

capacity, 

great 

dispersion, 

not much 

affected by air 



Fe 



1.947 



Mn 



Cr-V 



7.1 



25-35 



Weak 

penetration 

capacity, 

great 

dispersion 



90 



Mineralogical Analysis 



4.2. Qualitative diffractometry 



4.2.1 Overview of Preparation of the Samples 



See Fig. 4.3. 



Extraction 
of clay 

I 

<2 (im - 



Coarse 2-0.5 |um 
-► Medium 0.5-0.2 (im 
Fine < 0.2 \xm 



Plastic transfer_ 
paper 




Chemical 
or thermal " 
treatments 



Wet 
sample 

i 

Homogenization 
or analysis by level 



Air dry 
sample 



Centrifugation 
clay flake 



Oriented on Moderate 
plastic film grinding 

i 

Random 
powder 



^Homogenization 



. Agitate 
with water 



Oriented Oriented 
aggregates paste 



Porous 
plates 



Slides 

Whole Solvated Saturated Heated 

sample sample sample sample 

Basal Interlayer Lattice 



distance Expansion 



contraction 



Air dry 

I 
Moderate 
grinding 

i 

Random 
powder 



Fig. 4.3. Preparation of samples for X-ray diffractometry 



4.2.2. Preparation for Powder Diagrams 



Objective 

This method is a general way to identify mineral species without 
preferential orientations. It enables quantitative analysis as the 



X-ray Diffractometry 91 

relative intensity of maxima diffractions is approximately proportional to 
the number of crystals present if the level of anisotropy is low. 

Principle 

In the method based on the "spectrum of random powder" (Thomson 
et al., 1972; Peterson et al., 1986; Decarreau, 1990), the crystal is 
examined in the form of a fine isotropic powder under a monochromatic 
X-ray beam. Random orientation must statistically represent all possible 
orientations of the different particles and provide a complete spectrum of 
minerals likely to diffract the X-ray (clays, oxides, hydroxides, 
oxy hydroxides, non- weathered primary minerals, various salts, etc.). 

Each particle is considered to be a micro-crystal or an assembly of 
micro-crystals and the powder mass can be compared with a single 
crystal turning not in only one axis but in all possible axes. The powder 
spectrum makes it possible to fix the relative intensities of the peaks 
indexed by JCPDS and for this reason it should always be carried out 
before any other operation. 

After compression in the support at the semi-microscale, the "powder" 
sample includes micro-aggregates of fractal dimensions with a piled-up 
structure with open porosity - the grains being only very slightly 
connected - and its characteristics depend on the crushing, compression 
and homogeneity of the medium. The total average orientation at this 
scale is very weak. 

On the other hand, at the sub-microscale (a few tens or hundreds of 
nanometers), the primary morphological units are mixed stacks of clayey 
crystallites and can consequently present an orientation with a varying 
degree of disorder depending on the types of clay present. The use of a 
revolving support improves the level of randomization, but requires a 
relatively large quantity of powder, i.e. approximately 400 mg. 

Procedure 

A powder diagram can be performed on whole soil or on soil fractions 
(cf. Chap. 3). Clay obtained by centrifugation (centrifugation pellet or 
plastic transfer paper) is air dried and then crushed in an agate mortar to 
obtain a homogeneous powder: 

-With a spatula place in a hollow support (Fig. 4.4) the quantity of 
powder needed to almost fill the cavity. 



3 JCPDS - ICDD = Joint Commitee on Powder Diffraction Standards - International 
Center for Diffraction Data, Newtown Square Corporate Campus, 12 Campus 
Bdv, Newtown Square Pennsylvania 19073-3273 (USA). 



92 



Mineralogical Analysis 



- With a ground glass slide, gently flatten the powder 

- Gradually add powder to fill the remaining cavity and pack gently to 
bring the surface of the powder up to the reference plane 

Observations 

Randomization 

The powder must be sufficiently compact to provide cohesion without 
using a binding agent or needing to smooth the surface. Too much 
pressure can cause orientation. It is thus necessary to exploit the degree of 
"randomization-orientation" by preparing the powders as regularly as 
possible. Indeed, as the clay layers are planes, they tend to be oriented, 
which is likely to give irregular results. 

This effect can be limited by using a binding agent that is inactive both 
with respect to the X-ray and the sample (acetone, lake gum + alcohol, 
collodion + acetate of butyl or amyl, gum tragacanthe, etc.). These 
treatments make the powders more stable. However, the use of a vertical 
goniometer prevents separation of the powder when it is placed in the 
beam which makes this kind of preparation unnecessary in the majority of 
the cases. 



Fig 4.4. Supports hollowed out for 
diffraction: plastic support (a), 
Siemens revolving support (b), 
hollowed glass support (c) 




When only a small quantity of sample is available, double-sided 
adhesive Scotch tape can be used; this is powdered with the sample (the 
surplus can be removed by light tapping) or better still, by placing the 
sample on a silicon support. 

Freeze-drying makes it possible to obtain less oriented samples 
(although there is a risk of a few packages of layers displaying residual 
orientation). Rotating the sample enables maximum possible reflection, 



X-ray Diffractometry 



93 



which decreases the risk of error by improving randomization and by 
limiting fluctuations in intensity due to an insufficient number of 
particles. The rotating support requires larger quantities of powder. 

Granulometry - Focusing 

The use of a sample plane is required for satisfactory focusing of the 
beam. The roughness of the surface has a marked effect on the relative 
intensities of the lines. If the surface is rough, as is true in the case of a 
coarse powder, the absorption coefficient will be high and the intensities 
at small angles will consequently be exceptionally low. The powder must 
be fine (a particle size of about 10 |um) to avoid such fluctuations in 
absorption, but not too finely ground to avoid an artificial increase in 
amorphous minerals: 

- at 10 |um, fluctuations will not exceed 2% 

- at 50 |um, fluctuations can reach 20%. 

On the other hand, with a particle size lower than 0.02 urn, diffractions 
are diffuse and the intensity decreases. 

The width of the diffraction curve increases with a decrease in the 
thickness of the crystal. The structure of "amorphous" substances is 
characterized by the absence of periodicity or by short-range organization. 
In the latter case, they show only one statistical preference for an inter- 
atomic distance and XRD cannot give satisfactory results but only broad 
and badly defined peaks (Fig. 4.5). 



Intensity 



Fig. 4.5. Diffraction diagrams typical of 

(a) a well crystallized substance and 

(b) an amorphous substance with short- 
range organization 




Angle of diffraction 2& 



Particular uses of Powder Diagrams 

A powder diagram can be used to identify non-transformed primary 
minerals (quartz, calcite, etc.) or oxides and hydroxides of iron and 



94 Mineralogical Analysis 

aluminium, etc, as the degree of crystallinity in the case of kaolinites and 
the "fire-clays" will be distinguished more clearly: since the mode of 
stacking of the layers is different, certain peaks of kaolinite do not exist 
in "fire-clays", which may consequently not be seen on a oriented 
diagram. 

The di- or tri-octahedral nature of the layers can be highlighted by 
using ray 060 [filling of octahedral cavities by bivalent (1.54 A) or 
trivalent (1.49 A) cations]. 

Polymorphous illites can provide data that are characteristic of the 
mode of formation. 



4.2.3 Preparation for Oriented Diagrams 



Oriented Diagrams on Glass Slides 

Objective 

The objective is to identify certain clayey minerals capable of being 
oriented and to observe basal variations by means of heat or chemical 
treatments. The number of treatments needed depends on the mixture of 
minerals in the sample. 

Principle 

In diagrams of oriented aggregates, special weight is given to the 
crystalline planes parallel to the surface of the layers. The preferential 
orientation of silicates causes an increase in the maximum of basal 
differentiation J(001), which makes it possible to detect small quantities 
of crystalline species present in the mixture. 

With this method better diffraction is obtained of the species and 
fluctuations that are theoretically lower than with randomized powders 
since the particles do not exceed 2 |um. On the other hand, preferential 
orientation decreases the number of planes (hkl) in a position of 
diffraction. 

Procedure 

(a) Starting from a well-homogenized clay paste obtained by 
centrifugation, remove a small aliquot (approximately 400 mg) with the 
spatula and place it in a tube with 5 mL water; agitate to suspend 
particles. 

(b) Starting from a powder (for example recovery of the clay used in 
Chap. 9), weigh approximately 200 mg of clay, place it in a plastic tube 



X-ray Diffractometry 95 

with 5 mL water and a 8 mm glass ball and agitate for 2-3 min to 
disperse the clay, 
(c) Starting from a or b, pipette approximately 1 mL of suspension and 
spread it evenly on a 24 x 24 mm glass slide. The deposit must be 
spread evenly over the whole surface of the slide with no areas of extra 
thickness. If the deposit is too thin, there may be an effect of the 
support, if it is too thick, diffraction will occur only on the finest clay 
and the clay film may reticulate itself. Allow to dry at room 
temperature, then dry in the desiccator. Prepare three slides: 

- the first for examination of the rough sample without treatment 

-the second for examination of the sample after glycerol or glycol 
treatment 

- the third for heating the sample in the oven at 490°C (depending on 
requirements, this slide can be used for several thermal treatments at 
increasing temperatures). 

Observations 

After the suspension has been spread on the glass slide, micro- 
fractionation will occur as a function of the size of the clay particles since 
coarse clay sediments faster than fine clay. However, if drying is rapid, 
this segregation will not disturb qualitative interpretation. 

In contrast to powder techniques (cf. Sect. 4.2.2) that have to be 
sufficiently thick to limit the effects of orientation, oriented slides must 
be sufficiently thin so that the maximum number of basic units are 
suitably oriented. Plotting a black line on the support before use is a good 
way to judge the quality of the preparation: it should remain very slightly 
visible through the almost transparent clay film. 

In comparative semi-quantitative analysis, the suspended deposit of 
each sample should contain about the same quantity of clay. This is easy 
starting from dry clay which can be weighed before suspension. The 
removal of an aliquot of the sample during agitation results in slides with 
almost the same quantity of mineral material spread over the same area. 
From a suspension after agitation and homogenization, quickly remove 
an aliquot of the same volume and dry and weigh one of the samples to 
determine the concentration. 

For suspensions of wet materials, recover clay on a film of a given 
surface area and weigh an identical aliquot after drying. 

Amorphous minerals can mask part of the diagram. It is often 
necessary to eliminate them before recording the diffraction diagram. 



96 Mineralogical Analysis 

Diagram on Oriented Paste 

Principle 

This method is particularly suitable for minerals of the 2:1 type and 
halloysite-4H 2 0, the wet sample should be dried at room temperature and 
maintained at a relative humidity of approximately 80%. 

Procedure 

After insulation of clay to the required dimension by centrifugation (cf. 
Chap. 3), recover the wet centrifugation pellet, homogenize with a small 
stainless steel spatula, deposit it on a hollowed support (Fig. 4a), spread it 
out in the cavity, then smooth it with a glass slide to a perfect plane 
suitably located on the reference plane. Allow to dry slowly at ambient 
temperature taking care to avoid excessive desiccation. 

Observations 

On coarse 2 |um clays, the clay paste needs to be homogenized after 
centrifugation, as the part near the surface is able to concentrate fibrous 
clays which do not respect Stokes law. Smectites deposit preferentially at 
the surface whereas chlorites and illites sediment more quickly at depth 
and accumulate more extensively at the base of the centrifugation pellet. 
The same is true for iron oxides (density 4.5-5) which are heavier than 
aluminium oxides (density approximately 3). 

During drying, clays with a high coefficient of retraction can fissure and 
detach. A binding agent can be added, though there is a risk of 
introducing a variable that is not easy to control (disturbed orientation, 
flocculation, binding agent not amorphous for X-ray, etc). 

Specific Uses 

In certain cases, it is possible to work on the wet sample. With 2:1 clays, 
saturating the wet paste with Na + or Li + makes it possible to observe the 
phenomenon of unlimited swelling d(00\) > 30 A. During air drying, clay 
again reaches values of d (001) = 18, 15, 14 A. 

Aggregates Oriented on Porous Ceramic Plate 

Objective 

To allow retention of minerals that do not adhere on glass slides and/or, 
to allow successive treatments on the same sample: original sample, 
cation treatment, polyalcohol treatment, followed by successive heat 
treatments. This preparation enables satisfactory orientation of clays, and 
is particularly useful if the spectra are to be exploited for quantitative 
analysis. 



X-ray Diffractometry 



97 



Principle 

The clay is fixed on a porous plate by suction using a vacuum pump or by 
centrifugation using a Poretics ceramic porous disc and a Hettich support. 

Procedure 

Transfer the clay suspension on a 24 x 24 mm porous plate placed on a 
Buchner with a diameter of 40 mm whose bottom of sintered glass has 
been modified to create a 22 x 22 mm window, which is the only 
permeable part (Fig. 4.6). Tip the homogenized liquid onto the plate and 
apply a vacuum using a standard pallet pump. After formation of a thin 
continuous layer of clay, dry the sample and analyse by X-ray diffraction. 

Observations 

This medium and procedure gives well-oriented deposits with a reduced 
fractal dimension of the surface which is preserved after drying and is 
thus of excellent quality for XRD measurement. The thickness of the 
deposit must be homogeneous all over the plate and sufficiently thick to 
avoid the effects of the support, but thin enough to preserve a certain 
degree of elasticity and to avoid cracking caused by the rheological 
properties of the medium. 



Fig. 4.6. Preparation on porous ceramic 
plate 




Porous 
plate 



Modified 
sintered 
glass base 



Vacuum 



Measurements on porous plate make it possible to avoid excess 
solvation liquid and also to carry out the treatments required: chemical 
saturation by Mg 2+ or K + treatment, heating, etc. 0.20 |um millipore filters 
on glass plates can also be used for measurements (collection of 
suspended materials in water, airborne dust, etc). 

The cleaning of the porous plate is delicate and should be done after 
washing by slight abrasion of the surface that has been in contact with the 



Mineralogical Analysis 



clay. It is important to preserve the perfect flatness of the plate and to 
make sure contamination has not occurred. 



Oriented Aggregates Deposited by Ultracentrifugation On 
Semi-Flexible Film 



Objective 

The aim of this method is to obtain samples of the same thickness, the 
same mineralogical composition and the same apparent particle size. 

Principle 

Particles are deposited by means of a Sharpies super-centrifugal machine 
equipped with a cylinder with a semi-rigid internal plastic film (cf. Chap. 
3). The speed is regulated to collect particles of a known average 
diameter (coarse to fine clay). If necessary, the film can be analyzed at 
eight successive levels of 24 mm, which makes it possible to monitor 
modifications in the nature of clays as a function of particle size and 
density. To ensure the film is not too thick, the proportion of coarse, 
medium or fine clay must be known, and only the zones that correspond 
to an optimal density used (test of the black line on the film, cf. section 
"Oriented Diagrams on Glass Slides). 



Plastic film 
Sample support 

Stop 

Reference 
plane 

Homogenous 
zones 



1 




24 

< — ► 






































^ 


sj 
















O 
C\l 

1 












































' 


150 




1 
















Spring 



Glass 
plate 



Fig. 4.7. Diagram showing how to cut up a plastic film after continuous flow ultra- 
centrifuge and how to set it up for XRD. 



Procedure 

Remove the plastic film from the cylindrical bowl and dry flat for 
approximately 1 h. When it is still slightly wet and not yet rigid, cut it 



X-ray Diffractometry 99 

into sections following the 24 x 24 mm pattern drawn on the back of the 
film (Fig. 4.7). Each level should be labelled. Assemble the 24 x 24 mm 
semi-rigid film on a glass plate and place it on a support against a stop 
corresponding to the reference plane. 

Observations 

XRD can be performed directly on the oriented sample or after treatments 
(cf. Sect. 4.2.4) with polyalcohol; heating treatment at 110°C is also 
possible depending on the nature of the plastic used. The surface of the 
plastic support must be unpolished naturally without additives as these 
could cause background noise that is incompatible with the smoothness 
of measurements. 

4.2.4 Pretreatment of Clays 



Effect of the Pretreatments 

Different types of pretreatments can be used to facilitate the identification 
of clayey minerals by causing selective changes in the inter-reticular 
distances of the clayey layers. These treatments and their effects are 
summarized in Table 4.4. Table 4.5 shows the maximum inter-reticular 
distances observable in the clayey minerals of soils. 

Saturation by Cations 

Principle 

Saturation by a cation makes it possible to fill the existing cation 
vacancies and, by displacement of the exchangeable cations, to obtain 
homoionic samples that present uniform expansion of the layers of the 
expansible phyllosilicates (the quantities of interlayer water depend on 
the exchangeable cations). The divalent cations, e.g. Mg 2+ , Ca 2+ , Sr 2+ , 
give hydrates with two layers that are relatively stable in a broad range of 
relative humidity and are not very sensitive to the influence of hydronium 
ions during washing with water. 

Mg 2+ saturation gives a stable complex with two layers of inter-layer 
water which brings the expansible phyllosilicates to d00\ > 14 . Expansion 
of the layers allows differentiation of the non-expansible varieties of 
clays whose interlayer space is approximately 10 A (Table 4.5). 

iC saturation restricts interlayer adsorption of water and allows 
differentiation of 2:1:1 clays of the chlorite type, and of 2:1 clays of the 
vermiculite type. Non-expansible chlorites are not modified by this 



100 Mineralogical Analysis 

treatment, whereas vermiculites break down at 10 A. The same sample 
can be also used for smectites after heating to 550°C. 

Li + saturation followed by dehydration to approximately 300°C, 
followed by solvation with glycerol, makes it possible to differentiate 
montmorillonite from beidellite using the Greene-Kelly test based on the 
Hofmann-Klemen effect. When a smectite saturated with Li is 
dehydrated at 300°C, the Li interlayer migrates towards the octahedral 
layers which have a deficit of positive charges resulting from 
substitutions. The structure becomes non-expansible and there is no 
further inflation of the sample with glycerol treatment (9.5A). A 
beidellite (or a saponite) whose charges comes from tetraedric 
substitutions is not affected by such treatments and inflates with glycerol 
(17.7 A). 

Procedure for Mg 2+ saturation 

- Take an aliquot of 125-250 mg of clay 

- put in suspension and acidify with diluted hydrochloric acid to bring it 
to pH 3.5-4.0 to avoid precipitation of magnesium hydroxide 
(particularly if initial dispersion was carried out in an alkaline medium) 

- add 5 mol L _1 magnesium acetate solution to obtain a suspension of 
approximately 0.5 mol (Mg) L _1 

- leave in contact for 30 min 

-centrifuge for 5 min at approximately 3,000g and discard the 

supernatant 
-wash the centrifugation pellet twice with 0.5 mol (Mg(OAc)2) L _1 

solution to eliminate H + from the acid suspension then twice with 0.5 

mol (MgCl2 ) L" 1 solution(approximately 10 mL) 

- centrifuge and wash with 50% methanol (approximately 10 mL) then 
with 98% methanol (approximately 10 mL); then with 85% acetone. 
The silver nitrate test for CI must be negative 

- dry at room temperature for the powder spectrum, or prepare the plates 
for oriented spectrum immediately by adding a little water. 

Procedure for hC saturation 

- Take an aliquot of 100-250 mg of clays 

- put in suspension and add 1 mol (KC1) L _1 solution 

- centrifuge the flocculated clay after 30 min of contact 

- discard the supernatant 

- wash the centrifugation pellet with KC1 1 M 



X-ray Diffractometry 



101 



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102 Mineralogical Analysis 

Table 4.5. Maximum inter-reticular distance of soil minerals saturated with Mg 2+ 
and K + or solvated by glycerol treatment. 



mineral 




cf(A) 


hkl plane 


(a) Samples saturated by Mg 2+ 






chlorite 




13.6-14.7 


001 


vermiculite 




14.0-15.0 


002 


montmorillonite 




14.0-15.0 


001 


mica (lllite) 




9.9-10.1 


001 


talc 




9.2-9.4 


002 


halloysite 




10.1 


001 


metahalloysite 




7.2-7.5 


001 


kaolinite 




7.1 -7.2 


001 


lepidoscrocite 




6.27 


020 


boehmite 




6.11 


020 


gibbsite 




4.85 


002 


silicates 




4.4-4.6 


110 


gypsum 




4.27 


121 


goethite 




4.18 


110 


Cristobal ite 




4.04 


101 


lllmenite 




3.73 


102 


quartz 




3.34 


101 


feldspar 




3.1 -3.25 




calcite 




3.03 


100 


hematite 




2.69 


104 


magnetite 




2.53 


311 


trioctaedric silicates 




1.54 


060 


dioctaedric silicates 




1.49 




(b) Samples saturated by Mg 2+ and solvated by glycerol 


montmorillonite 




17.7 


001 


vermiculite 




14.4 


002 


chlorite 




13.6-14.7 


001 


halloysite 




10.1 -10.7 


001 


montmorillonite (2nd order) 


9.5 


001 


chlorite- Vermiculite 


(2nd order) 


7.15 


002 


minerals giving inter- 


layer spaces 


similar to 'a' 





X-ray Diffractometry 103 

(c) Samples saturated by K + and air dried 

chlorite 13.6-14.7 001 

montmorillonite 11.0-13.0 001 

vermiculite 10.0-11.0 002 

metahalloysite 7.2 - 7.5 001 

chlorite (2nd order) 7.15 002 
Minerals giving inter-layer spaces similar to 'a' 

d) Samples saturated by K + and heated at 550°C for 2-3 h 

chlorite 13.6-14.7 001 

montmorillonite 9.9 - 10.1 001 

vermiculite 9.9-10.1 002 

mica (lllite) 9.9-10.1 001 

chlorite (2nd order) 7.15 002 

- then wash with 50% and 95% methanol and finally with 95% acetone 
until there are no CI" ions in the washing solution (no precipitate with 
addition of AgNC^) 

- allow to dry at room temperature or immediately prepare slides for 
oriented spectrum by adding distilled water. 

Procedure for Li + saturation 

- Take an aliquot of 1 10 - 250 mg of clay 

- put in suspension and add 3 mol (LiCl) L _1 solution 

- leave in contact for 30 min 

- centrifuge and discard the supernatant 

- rapidly wash the centrifugation pellet with 1 mol (LiCl) L _1 solution 
then with a little water 

- make an oriented slide and dry at 250°C overnight 

- carry out the glycerol treatment (1 night) and perform the diffraction 
spectrum. 

Remarks 

- This test is not completely selective 

- mounting on an ordinary glass slide can cause errors during heating as 
the Na + in glass can exchange with the Li + in the clay, which causes 
incomplete neutralization of the octahedral charges 

- the glycerol treatment must be carried out hot for a period of several 
hours to allow complete expansion of the layers 

- an irrational basal sequence indicates inter-stratification. 



104 Mineralogical Analysis 

Removal of Iron 

Principle 

Iron often has to be removed first to mitigate its action on the process of 
measurement by XRD using a Cu tube (X-ray fluorescence increases 
background noise) and second to avoid dilution through a reduction in the 
intensity of diffraction. 

Different methods can be used to eliminate the different forms of iron. 
These methods vary in vigour and should not transform the phyllo- 
silicates present, but make it possible to complex and reduce amorphous 
and crystallized iron in a slightly acid medium with the minimum 
possible degree of aggressiveness. These methods are similar to those used 
in Chaps 2 and 6, but the solubilization of iron compounds is generally 
not controlled (because it is the final residue of the sample will be analyzed 
by XRD). 

It should be noted that the amorphous silicon-iron complexes present 
in certain sediments will be dissociated in an acid medium giving soluble 
iron and precipitated amorphous silica. 

Hematite and goethite oxides are only slightly affected by an acid 
oxalate treatment at pH 3. A reducing treatment with complexation of the 
products of dissolution (oxalate acid + dithionite) is the best way to 
eliminate most iron while sparing the clay. Oxalate dissolves amorphous 
iron and dithionite dissolves oxide forms of iron. A slightly acid medium 
allows extraction of "free iron" but at the same time extracts part of the 
iron of the lattice of certain clays, for example vermiculites. With 
dithionite, the presence of sulphur precipitated after reduction does not 
pose a problem for XRD, except for a dilution effect on the sample. 
Sulphur should be eliminated from the extraction pellet after 
centrifugation and drying. 

Procedure 

The main methods are based on dissolution in an acid or base sequestering 
medium and/or reducing medium (cf Chap. 6): 

- The DCB method (dithionite, citrate, bicarbonate) extracts iron from the 
majority of its amorphous and crystalline compounds by reduction and 
sequestration without significant modification of aluminosilicates or 
lithogenic hematite 

- the method based on acid oxalate at pH 3.0 (in darkness, or with UV 
photolysis) extracts noncrystalline forms 

-the sodium pyrophosphate method at pH 9-10, EDTA at pH 9-10, 
acetyl acetone extracts organometallic forms 



X-ray Diffractometry 105 

-the tetraborate method at pH 8-9 extracts Fe from monomeric 

complexes. 

The elimination of iron is accompanied by the dissolution of aluminous 
products, silica, etc. which can be controlled by chemical titration of the 
extracts. 



Solvation Treatments 

Principle 

Solvation by polar molecules, such as mono or polyhydric alcohols, 
ethers, amines, results in the formation of interlayer organic complexes. 
The resulting structure is more stable than the structure of dehydrated 2:1 
clay. Swelling is all the easier since the charge of the layers is weaker, or 
is limited to the octahedral layer. The nature of the interlayer cations 
modifies the limits of stability of the organic complex. The basal distance 
of the smectites reaches 17.7 A with a double interlayer layer of glycerol, 
and 17.1 A with ethylene glycol. The rate of hydration can vary 
considerably. Montmorillonites inflate more easily than the majority of 
clays. 

Impregnation can be accomplished in the liquid or vapour phase by 
heating to 60°C. In certain cases, the treatment has to be continued for 24 
hours to take the slowness of interlayer expansion into account. 
Condensation of the vapour does not cause any mechanical disturbance 
and gives more intense lines of diffraction. 

Procedure for Glycerol Treatment 

This procedure is based on that of Modre and Dixon (1970): 

- Prepare a 1 : 10 mixture of glycerol and water 

- on a previously air-dried oriented plate, apply a film of glycerol in a 
very fine spray, taking care not to create an excess of reagent 

- allow to dry for at least 1 or 2 h and then perform XRD. 

Caution. The complex loses its effectiveness over time, so it is 
advisable not to wait more than 20 h. Using ceramic plate for this 
treatment makes it possible to eliminate excess glycerol. 



106 Mineralogical Analysis 

Procedure for Ethylene Glycol Treatment 

r— i 



Vacuum 



i 




Sample 



— — — — I Ethylene glycol 



Fig. 4.8. Treatment of the samples with ethylene glycol 

This procedure is based on that of Eltantawy and Arnold (1974) and 
Chassin(1974): 

-place the sample (generally for spectrum-oriented aggregates) in a 
desiccator containing the ethylene glycol (Fig. 4.8) 

- create a partial vacuum with the vacuum pump and leave the samples in 
contact with the vapour phase for at least one night 

- remove the sample and perform XRD as soon as possible. 

Caution. The complex loses its effectiveness over time, so it is advisable 
not to wait more than 10 h. 

Safety. Ethylene glycol or 1,2 ethanediol: HOCH 2 -CH 2 OH, has a boiling 
point of 196°C; it is hygroscopic, toxic by ingestion, and can affect the 
kidneys, lungs and the heart. 



Intercalation Complexes 

Principle 

Intercalation complexes are very useful particularly to separate 1 : 1 clays 
and to distinguish well-crystallized forms, disordered forms and 
halloysites. Since the forms of these species are similar, it is impossible to 
separate them with precision (kaolinite and halloysite can be found in 
flat, tubular or glomerular forms). One common procedure is first to form 
the intercalation complex, then after obtaining the spectrum, to move the 
complex with water and perform solvation with ethylene glycol or 
glycerol. The stability of the intercalation complexes is variable and XRD 
spectra should be performed without delay. 



X-ray Diffractometry 107 

Treatment with hydrazine hydrate (Wada and Yamada, 1968; Range 
et al. 1969; Calvert, 1984) allows the inter-layers of the well-ordered 
kaolinite to be changed from 7.15 to 10.48 A without modifying the 
chlorite. The presence of interstratified minerals can be awkward. 

In the treatment using dimethylsulfoxide (Gonzalez Garcia and 
Sanchez Camazano 1968 Olejnik et al., 1968 Anton and Rouxhet, 1977; 
Calvert 1984): (i) kaolinite forms an intercalation complex that increases 
the interlay er distance from 7.15 to 11.18 A, which remains stable after 
heating to 300°C, (ii) halloysite and dickite display identical behaviour, 
except that heating to 300°C does not result in further expansion, (iii) 
vermiculites and smectites increase from 18 to 19 A and (iv) chlorite 
undergoes no change. 

Metahalloysite forms an intercalation complex with formamide. The 
line rapidly reaches 10.4 A and whereas for kaolinite there is no reaction 
even after 4 h of contact (Churchman et al., 1984). 

Procedure for Hydrazine Hydrate Treatment 

-Place the clay sample on a glass slide or on porous ceramic in a 
saturator containing the hydrazine hydrate 

- create a partial vacuum with the vacuum pump and leave the sample in 
contact with the vapour phase at 65 °C for at least one night 

- remove the sample and perform XRD without delay. 

Remarks 

It should be noted that the complex loses effectiveness over time; so it is 
advisable not to wait more than 1 h before XRD. 

Sequential treatment with hydrazine + water + glycerol makes it possible 
to increase the basal interlay er distance of halloysite to 11.1 A, whereas 
that of kaolinite remains at 7.15 A. 

Safety 

- Hydrazine hydrate: H 2 NR-NH 2 ,H 2 0, boiling point 1 19°C 

- miscible with water and ethanol 

- strong base, very corrosive, attacks glass and the skin 

- highly toxic, causes irritation of the eyes, may be carcinogenic. 

Procedure for Dimethylsulfoxide Treatment 

- Put an aliquot of clay weighing from 100 to 250 mg in suspension in 5 
mL of dimethylsulfoxide 

- leave in contact for 8 h in a water bath at approximately 40°C 

- agitate from time to time 

- centrifuge and prepare slide, allow to dry 

- rapidly perform XRD after drying. 



108 



Mineralogical Analysis 



Table 4.6 


l. Effect of the thermal treatment on 1 


the diffraction of clays (TDA 




= characteristic temperatui 


r e of change in thermal differential analy- 




sis, cf. Chap. 7). 












temp. (°C) 




mineral 




4 H 
at 


TDA 


heat effect 




Well crystallized 
Kaolinite 


500 


575-625 


absence of diffraction 




Disordered kaolinite 


500 


550-562 


absence of diffraction 


1:1 


Dickite 


500 


665-700 


absence of diffraction 


clays 


Nacrite 


500 


625-680 


absence of diffraction 




Halloysite, 4H 2 


110 


125-160 


elimination of water 






500 


560-605 


absence of diffraction 




Metahalloysite, 2H 2 


110 


125-150 


elimination of water 






500 


560-590 


absence of diffraction 


Short- 
range 


Allophane 


110 


140-180 


elimination of water, no XRD 
spectrum 


order 
alumino- 


Imogolite 


350 




difficult identification by XRD 
^18A 


silicates 




500 


500 


disappearance of 18A peak, 
destruction of the mesh 




Crystallized micas 
(muscovite) 


490 


700 


mica spectrum up to 1 ,000°C 






350 


125-250 


loss of water 




lllite-micaceous clays 


500 


350-550 


mica spectrum 






500 


700 


mica spectrum (001) 


Micas 


Glauconite 


500 


530-650 


loss of water-mica structure 


and 


Celadonite 


500 


500-600 


mica structure 


2:1 


Biotite 


500 


700 


mica spectrum until 700- 
1,000°C 


clays 


Vermiculite 


300 


300 


Progressive loss of H2O 
the initial basal space (001) 
is a function of moisture: 14- 
13, 8-11, 6-9A changes 




Montmorillonite 


300 


300 


Disappearance 15 -> 9A 



X-ray Diffractometry 



109 



Chlorite 



Chlorite-Mg 



2:1:1 



Clays 



Chlorite-Fe 



Chlorite-AI 
Inter-stratified 



500 680-800 no change if structure is well 
ordered 



500 650 



500 500 



intensification of line 14 A - 
line 3.54 A not affected 
(octahedral layer 820°C 
intercalated layer 640°C) 

Attenuation of line 14A which 

becomes broad and diffuse. 

octahedral layer 530°C, Fe 2+ 

intercalated layer 430°C, 

Fe 2+ 

intercalated layer 250°C 

Fe 



3+ 



500 



500 
<600 



(octahedral layer 750°C 
intercalated 900°C layer) 
effects vary with the mineral 
species present 



Fibrous Sepiolite 



Clays Palygorskite 



350 



300 
500 



<200 
>200 
350 
800 

<400 

440 

800 



dehydration 

space 12 A becomes weak 

and diffuse -» 9.8 A, space 

7.6 A more intense 

recrystallization 

dehydration without change 
in structure, 10.5 A peak 
becomes broad and diffuse, 
destruction of structure 



Safety 

- Dimethylsulfoxide: C 2 H 6 OS, boiling point: 189°C 

- hygroscopic, irritation of the skin (urticant), keep away from the eyes. 

Procedure for Formamide Treatment 

- Place the clay sample on a glass slide or preferably on porous ceramic, 
allow to dry 

-vaporize formamide and note the time of application; when the 
formamide excess has been eliminated (approximately 20 min), perform 
XRD (repeat with the same sample at the end of the day and compare 
the two spectra). 



110 



Mineralogical Analysis 



Safety 

Formamide (H-CO-NH 2 ) is an ionizing solvent which can release a 

slight odour of ammonia. It dissolves lignin. No known risk to health. 

Thermal Pretreatments 



Principle 

The hydrated minerals undergo modifications due to the effect of the rise 
in temperature. These modifications occur at certain characteristic 
temperature stages. The length of time at a given temperature is also 
significant. The transformations take place with varying rapidity 
depending on the nature and the degree of crystallinity of the thermally 
sensitive minerals. In general, 2-4 h are required 

Table 4.7 Effect of thermal treatments on oxides and hydroxides of aluminium 
and iron (TDA: characteristic temperature of change in thermal diffe- 
rential analysis, cf. Chap. 7) 

mineral temperature (°C) heating effect 

4H TDA 

iron series 
spectrum of disordered hematite 
spectrum of we 1 1 -crystallized hematite 
spectrum of disordered hematite 
spectrum of we 1 1 -crystallized hematite 
yFe 2 3 broad peak 

-» hematite spectrum 
-^ hematite spectrum 
-» hematite spectrum 
At 300°, the akaganeite spectrum weakens 
gradually 

-^ hematite spectrum 
-» hematite spectrum with intermediary 
goethite 

unstable in air at ambient temperatures 
goethite spectrum after air drying 
-^ hematite spectrum 



Goethite 


300 


230-280 




900 


900 


Goethite 


350 


240-350 


alumineuse 




900 


Lepidocrocite 


300 


230-280 
400-500 


Maghemite 


350 


350^50 


Magnetite 


500 


600-800 


Akaganeite 


300 


200^00 
420-500 


5FeOOH 


300 


140-260 


Feroxyhyte 






5FeOOH 






Ferrihydrite 


350 


350-400 


(without Si) 







X-ray Diffractometry 



111 



Ferrihydrite 
(with Si) 



500 



550-600 -^ hematite spectrum 



Aluminium series 



Gibbsite 


200 


150-200 


Bayerite 


200 


150-200 


Nordstrandite 


200 


150-200 


Boehmite 


500 


450-500 


Diaspore 


500 


470-500 


Procedure 







-> boehmite and yAI 2 3 spectrum/a? 

-^ boehmite and yAI 2 3 spectrum/a? 

-> bayerite spectrum -^ boehmite 

^yAI 2 3 spectrum 

-> spectrum of disordered corundum towards 

spectrum of crystallized corundum 



Place the samples assembled on glass slides in a cold furnace; bring the 
furnace to the desired temperature (110, 350, 490, 530°C, etc.) and 
maintain the temperature for at least 4 h. Heating must be progressive to 
avoid breaking the glass slides and possible reticulation of the clay film. 
The furnace should then be allowed to cool gradually, opening the door 
to accelerate the process if necessary. If they have not undergone 
irreversible transformations, the slides can be stored in the desiccator 
until XRD. Table 4.6 shows the influence of heat treatments on the 
diffraction of clays. Table 4.7 shows the influence of heat on the 
diffraction of aluminium oxides and iron hydroxides. 

Observations 

-Loss of interlayer water results in contraction of the mesh and 

displacement of the basic diffraction lines 
- heating to high temperatures can lead to collapse of the lattice and 

dispersal of the characteristic X-ray spectrum. 

Example 

Dissociation of kaolinite by heating (Fig. 4.9) to around 490°C can be 
visualised by the four following reactions: 

(1) at 500°C: Al 2 3 ,2Si0 2 ,2H 2 -> Al 2 3 ,2Si02 (metakaolinite) + 2 
H 2 

(2) at 925°C: Al 2 3 ,2Si02,2H 2 -> Al 2 3 ,Si0 2 (sillimanite) + Si0 2 
(6 quartz) + 2 H 2 

(3) at 1 100°C: Al 2 3 ,2Si0 2 ,2H 2 -> aAl 2 3 + 2Si0 2 (6 quartz) + 
2H 2 

(4) at 1 400°C: Al 2 3 ,2Si0 2 ,2H 2 -> 1/3 (3Al 2 3 ,2Si0 2 ) + 4/3 Si0 2 
(6 quartz) + 2H 2 



112 



Mineralogical Analysis 



All these reactions are possible starting from 800 K (approximately 
527°C). There is no obstacle to the transition of kaolinite — > 
metakaolinite — > sillimanite — > mullites — > oxides, the most stable 
system, which depends on the temperature. Thermodynamically reaction 
(1) occurs first (Fig. 4.9). 



AGO, kcal mol 




TK 



Fig. 4.9. Transformation of kaolinite by heating 



Preparation of Iron Oxides forXRD 



Principle 

The use of XRD to study iron oxides in the soil requires a sufficient 
concentration of the different iron phases. Consequently the products 
have to be concentrated and purified without modifying either their 
crystallinity or the level of substitution by aluminium, and without 
causing chemical conversions of the phases. The following methods can 
be used to this end: 

- separation methods (concretions, separation by density gradient, 
magnetic separation, etc 

- in situ XRD determination on an uncovered thin slide, or extracted 
micro-samples with high iron content. 

Chemical methods only enable the concentration of iron, which can 
exist in the form of coating, in amorphous forms (ferrihydrite, etc.), 
or involve varying degrees of crystallization (goethite, hematite, 



X-ray Diffractometry 113 

lepidocrocite, etc.). Clay minerals are dissolved by 5 mol (NaOH) L _1 
solution under boiling. Kaolinites, halloysite, gibbsite, amorphous 
aluminosilicates, etc. are destroyed and solubilized, but 2:1 clays are 
more resistant and are thus only partially attacked. In addition to iron, 
smectites, illite, quartz, anatase, and rutile are also found in the residue. If 
the amount of silica present is not sufficient, during heating with 5 
mol (NaOH) L _1 solution, the ferrihydrites are likely to be transformed 
into hematite or goethite. If the sample is attacked using 5 mol (NaOH) 
L _1 + 0.2 mol (silica) L _1 solution, the rate of aluminium substitution is 
not significantly modified. 

Procedure 

-Weigh in PTFE beakers 2-5 g of soil, or granulometric clay <2 |um 
depending on the total iron content 

- attack with 20 mL 5 mol (NaOH) L _1 + 0.2 mol (silica) L _1 solution, 
cover the beaker with a PTFE plate and boil for one hour 

- allow to cool and decant 

- dilute the medium with deionised water, then wash on filter with water 
until the pH is neutral 

- perform XRD 

Note: a powder diagram is better than an oriented diagram for these 
products. 

Remarks 

-If the soil or clay contains many 2:1 phyllosilicates, the concentration 
may be too low; in this case it is useful to perform two differential 
spectra (DXRD), one with the original product, the other with the 
chemically concentrated product, or with the spectrum obtained after 
dissolution of iron by the citrate-bicarbonate-dithionite mixture (CBD, 
cf. Chap. 6) 

- a cobalt X-ray tube should be used to avoid fluorescence of iron which 
occurs with a copper tube 

- the PTFE beakers can be used up to a maximum temperature of 250°C; 

- in certain cases, destruction of the matrix silicates can be achieved 
through an HF attack in a PTFE beaker 

- heating to 600°C gives anhydrous oxides — > hematite. 

4.2.5 Qualitative Diffractometry 

The sample of whole soil crushed to 0.1 mm and "clay < 2 |um" fractions 
(or 0.5 or 0.2 |um sub-fractions,) prepared for randomized or oriented 
powder spectrum are analyzed by XRD. 



1 14 Mineralogical Analysis 

Each group of clay has its own layered structure which gives basal 
reflections whose position, intensity and shape enable either immediate 
identification or after specific treatments. This section describes the 
interpretation of standard situations that make it possible to evaluate 
clayey minerals at the scale of the group and the sub-group, and very 
occasionally at the scale of the species. 

Fine Adjustment of Experimental Conditions 

The experimental conditions must be precisely determined in advance, 
i.e. the type of tube (Cu, Co, etc.), the intensity applied to the filament, 
the opening of the slits, the speed of rotation of the goniometer, tension 
meter, amplification, adjustment of the constants of time-inertia, 
sensitivity, signal/background ratio, angular zone, scanning speed, etc. 

A cobalt tube X-ray should be used for samples that are rich in iron 
because it does not generate fluorescence with this ion. Generally, with 
suitable geometry, an international standard Cu tube give a satisfactory 
performance, and JCPDS tables and software (see note 1 in Sect. 
4.2.2) cited here are based on this standard. 

The rotation speed of the goniometer and the selection of the angular 
zone of scanning determine the time needed for analysis. The choice of 
the scanning rate of the sample influences the relative precision of the 
diffractograms. Too high a speed can lead to insufficient discrimination. 
For the determination standards, a displacement speed of 1 to 2 °min _1 
(29 angle) may be sufficiently rapid but does not allow adequate 
separation of fine doublets such as those of kaolinite (002) and chlorite 
(004) for which a slower rate is necessary (<0.5° 20min _1 ). For rapid 
characterization of clay (group and sub-group) the angular zone of 
scanning can be limited to a 29 zone of approximately 2-40 degrees, if 
determination of the d(060) diffraction is not required as this takes much 
longer. The strong reflection at 29= 60° makes it possible to estimate 
parameter "Z?" and thus to differentiate dioctahedral minerals (1.48-1.50 
A) from trioctahedral minerals (1.53-1. 55 A) and sometimes to provide 
more evidence for approximate values by bringing these values closer to 
the intensities of d(00l) diffractions, for example in the identification of 
certain micas. Powder samples are performed by rotating the sample- 
support in the reference plane, which requires high precision equipment. 
The oriented sample method makes it possible to reinforce the reflections 
(001) by directing the particles according to the development plane of 
clay minerals. All the slides (or any other form of support) for each 
sample should be made simultaneously and as homogeneously as possible 



X-ray Diffractometry 115 

(same suspension, same quantity of clay per slide, etc.). Three sub- 
samples are usually essential: 

- a reference oriented sub-sample without treatment, dried and observed 
with a known quantity of relative moisture 

- an oriented sub-sample solvated for hydrated 2:1 clays 

- an oriented sub-sample for heat treatment (500°C for 4 h). 

The samples should be analysed one after another, and the equipment 
should be regulated in exactly the same way for each sample to enable 
better comparison of the spectra. In more complex cases, it may be 
necessary to prepare homoionically saturated samples (K + , Mg 2+ ) with 
suitable treatment and intercalation sequences. These should be 
performed under the same conditions taking into account the relative 
humidity of the air, relative stability of the solvations and complexes of 
intercalation, reaction times (in the case of formamide treatment whose 
action is rapid on halloysite, whereas on kaolinite this treatment can take 
4 or 5 h and should thus be performed at the head of the series). If the 
sample is very small, it may be possible to perform treatments and 
measurements sequentially on the same sample. In this case porous 
ceramics should be used (or 0.20 |um Millipore filters if heat treatments 
are not envisaged). However, as measurements must then be made over a 
period of several days it is more difficult to maintain constant conditions. 

Examination of the Diagrams 

Background Noise 

The background noise is due to non-specific signals that are inherent to 
the material, to the process, to minerals with short-range organization, 
and possibly to fluorescence phenomena resulting from the emission of 
secondary X-rays. The latter phenomenon can be eliminated by the back 
monochromator and amplitude discrimination. The background noise can 
be also smoothed electronically with suitable software. In the zone of the 
small angles (29 between and 10° approximately), depending on the 
adjustment of the slits, the counter may receive part of the direct beam or 
of reflections from the edges of the support. The use of variable slits 
generally prevents these phenomena. 

The presence of amorphous substances (aluminosilicates, different 
oxides, etc.) is indicated by broad and diffuse bands. The interpretation 
software makes it possible to eliminate these zones selectively and to 
stabilize the base line. 



1 16 Mineralogical Analysis 

Geometry of the Peaks 

The geometry of the peaks enables determination of the degree of 
crystallinity, particularly for well-crystallized minerals where crystal size 
is suited to the X-ray wavelength and presents regular inter-reticular 
variations giving clear diffractions and many harmonics (Fig. 4.5a). 
Badly crystallized minerals that are not well ordered and whose size is 
not suitable to X-ray often give broad biconvex peaks whose surfaces are 
not usable for quantitative analysis (e.g. smectite, Goethite, see Fig. 
4.5b). Too many particles such as diatoms, quartz or feldspars, can limit 
the orientation of clays and render interpretation of the spectra difficult. 
Superposition of peaks resulting from the cumulative presence of several 
minerals results in a widening of the signals and an abnormal rise in 
relative intensities, for example Chlorite (OOl)-Vermiculite or Kaolinite 
(OOl)-Chlorite (002). These samples require different pretreatments to 
reduce uncertainty and to reveal the masked peaks (cf. Sect. 4.2.4). 

Peaks may display low intensity and widening even in the case of 
minerals that are usually well distinguished such as quartz, calcite or 
ordered kaolinite (001 or 002) if the sample is not thick enough. 
Goethites and smectites which have more diffused peaks may be masked. 
At certain angles, lines that come from the support are also likely to 
appear. 

Particles of approximately 0.5 |um are used for comparative studies on 
different clayey fractions and for quantitative analysis, when separation 
was done by ultracentrifugation. Widening at half-height of the peaks of 
other coarser or finer fractions must be controlled. Indeed, if the 
crystallites are too fine, the reflections widen; if they are too big, 
absorption phenomena can disturb the intensity. It should be noted that 
smectites are concentrated in the finest fractions; kaolinites, illites, 
chlorites, iron oxides in the 0.2-0.5 |um fractions; detrital micas, 
chlorites, quartz, feldspars in the 0.5-2 |um fractions. A well-crystallized 
but incompletely delaminated kaolinite can be found in the fine silt phase. 

Location of the Angular Distances 

The angular location of the top of the peaks of diffraction must be 
transformed into interreticular distance "J" in A, in accordance with the 
Bragg equation (1) (see table or software depending on the type of anti- 
cathode used). Each peak should numbered chronologically and its 
reticular distance given in Angstroms or nanometers (international 
standard), along with its relative intensity. Computer equipment allows 



X-ray Diffractometry 117 

on-screen comparisons with reference spectra and an automatic search of 
the JCPDS database (note 1 of Sect. 4.2.2). 

The powder spectrum of the whole soil provides an overview of all the 
minerals present and of the relative intensities of the different 
components. The spectrum may not be easy to interpret if this is not done 
by comparing it with other samples of the same sequence but at least 
makes it possible to select appropriate samples for a more thorough 
examination. The clay fractions of the spectrum powders, which are 
purified and concentrated by dispersion and particle-size separation 
treatments, provide more precise information on the clays and on 
associated minerals. It is possible to determine predominant peaks with 
strong intensity, and assemblies of characteristic peaks at the level of the 
reticular distances from 7-, 10-, 12-, 14-15 A, as well as the peaks of 
quartz, calcite, etc., and possibly the peaks at 4.40-4.50 A that are 
identifiable in the majority of clays. The same limitations apply to 
complex mixtures. The peaks are often broad, and the profiles 
asymmetrical; smectites only display a reproducible interlayer space if 
their state of hydration and ionic saturation are controlled. 

The presence of interstratified minerals can cause problems that are 
difficult to solve, especially for the di- and tri-octahedral sub-groups 
because of obstruction of the zone (060) around 1.50A. Depending on the 
objectives, a spectrum helps determine the strategy to be used e.g. 
adjustments of the experimental conditions, or of the number of oriented 
plates with given treatments, and possibly the need for other 
complementary techniques such as thermal analysis, IR spectrometry, 
electronic microscopy, selective dissolutions, chemical purifications, and 
so on. 

On oriented spectra that have undergone suitable treatments basal 
reflections, relative intensities, regrouping of the diagnostic reflections 
using JCPDS-ICDD interpretation tables (note 1 in Sect. 4.2.2), can 
be performed rapidly on-screen using suitable software. The table 
designed by Brindley and Brown (1980) can also be used, it takes into 
account the most intense lines characteristic of clay and associated 
minerals in soils, classified in descending order of the values of "J". The 
"Hanawalt Mineral Search Index" arbitrarily divides the field of the 
reticular distances from 999.99- to 1.00 A into 40 groups. The first entry 
corresponds to the line of maximum intensity. The value "d" of the 
second line corresponds to the second strongest intensity and determines 
the sub-group. The entries are then classified within each sub-group by 
decreasing values of intensity resulting in six additional lines. Since the 
degree of intensity is difficult to determine, multiple entries make it 



118 Mineralogical Analysis 

possible to integrate experimental variations: if the diffraction of a 
mineral includes 2 or 3 high-density peaks, there can be 2 or 3 entries 
(with intensities ranging from 100 to 75). The record of the location of 
the eight peaks should be followed by the name of the mineral and the 
number of complete JCPDS cards concerning this mineral. The behaviour 
of minerals under different chemical and thermal treatments facilitates the 
identification of clay minerals (see Tables 4.4 - 4.7). 

4.3. Quantitative mineralogical analysis 

4.3.1 Interest 

Quantitative mineralogical analysis is used to identify factors that 
influence or determine current or previous pedogenesis and physical and 
chemical properties of soils. This type of analysis is the logical 
continuation of qualitative analysis but many problems are involved and 
the precision will be influenced by the chemical and structural 
complexity of the substrate. For a simple substrate with two main 
mineralogical species, depending on the methods used, the risk of error 
may be around 3%, but may reach 5-10% with three species and about 
30% for mixtures with n species. The presence of substances with short- 
range order further increases the degree of inaccuracy and can even 
prevent determination with certain methods, if this is the predominant 
mineral form. The methods use either: 

- a single instrumental technique, XRD being the most widely used 

- a group of techniques that make it possible to identify the centesimal 
composition of the sample more satisfactorily, although with increased 
complexity and at a higher cost. 

4.3.2 Quantitative Mineralogical Analysis by XRD 
Principle 

The advantage of these analytical methods is their relative simplicity and 
especially the fact that a single instrumental technique is involved. X-ray 
intensities obtained for each component of a mixture are directly 
correlated with the proportion of the component according to the 
equation: 



X-ray Diffractometry 119 

where W v is the weight of compound P in the sample; I v is the intensity 
of the diffraction of the pure compound P; k ispthe constant depending on 
the compound P and the experimental conditions and |u is the attenuation 
coefficient of mass of the mixture. 

When the substances are well crystallized and present a definite 
chemical composition, resulting in intense specific XRD reflections with 
no superposition, quantification is easy and the degree of precision is 
acceptable, particularly with a standard giving the same chemical 
characteristics. Unfortunately, the imperfection of soil minerals (structural 
order and disorder), the size of crystallites, chemical variability due 
to substitutions, and preferential orientations result in modifications in 
the intensity of peaks for the same mineral and even in angular displacement 
(which makes the selection of reference minerals for the calibration of 
measurements difficult). In this case the resulting measurements are very 
precise. When possible, the reference minerals should come from the same 
geological formation and have undergone the same type of pedological 
deterioration in order to reproduce a matrix with a similar degree of disorder 
and chemical composition (clays under transformation processes). 

The samples for quantitative analysis have to be prepared with 
particular care to limit widening of the lines (over-grinding can result in 
an increase in structural disorder as well as in an increase in the 
amorphous phase), the effects of extinction and micro-absorption 
(crystallites and particles are too coarse), and phenomena of orientation 
due to the interrelationships between particles (preferential orientation). 
Measurements are taken on: 

-powder samples that account for all the reflections; as the relative 
intensity of the basal reflections is low, a concentration of 
approximately 10% may be necessary to quantify a given compound in 
the mixture; 
- an oriented sample which increases the basal reflections of clays, 
however, this involves the risk of certain components obstructing the 
regularity of the orientation; this technique is more sensitive and pushes 
back the limits of detection, but instead of preparing the sample on 
glass slide, the sample should be prepared on porous ceramic to 
eliminate the effects of sedimentation (cf. Sect. "Aggregates Oriented 
on Porous Ceramic Plate"). 

Sample preparation can include Mg 2+ saturation, solvation with 
glycerol and finally, mixing an internal standard and a matrix suppressor. 
The homogenization of the sample and the internal standard should be 



120 Mineralogical Analysis 

carried out in a mixer with an agate ball. When clay samples are 
separated by ultracentrifugation, a check should be made to make sure 
there are no variations in composition (the relative proportion of each 
clay in the mixture may be different and cause bias). Three types of 
quantitative XRD methods can be used: direct analyses without standard, 
analyses with an external standard, analyses with an internal standard 
(addition of standard, matrix suppressor, etc.). 

In direct analysis with no standard the mineral of reference is taken 
directly in the sample matrix. Interesting relative values will be obtained 
for the comparison of samples from the same sequence. 

Analysis with an external standard does not require long preparation, 
but the choice of standard substances for the calculation of the intensities 
is difficult as is also the case with the other methods. The degree of 
precision is thus sometimes doubtful except for compounds that give 
clear reflections in a relatively pure medium with only few major 
components, for example well-crystallized kaolinite and quartz. 

Analysis with an internal standard: these analyses are carried out in the 
presence of a standard substance presenting (i) a low attenuation 
coefficient, (ii) a strong XRD reflection that is narrow and is not 
superimposed on reflections of the minerals to be determined, and, if 
possible, is low in harmonics and (iii) a density that is close to the 
minerals in the mixture enabling better homogenization. 

The aAl 2 3 corundum was adopted in 1976 by JCPDS (see note 1 in 
Sect. 4.2.2) as the standard of reference for the quantitative study of 
minerals (high-purity synthetic corundum, 1 |Lim particle size, Linde, by 
Union Carbide or similar). The basal reflection at 2.085 A (/= 100 for the 
1,1,3 plane) is clear. Crystallinity can be increased by heating at 800°C 
for 1 h. There is no preferential orientation. The 2.106 A line of MgO, 
and the 6.1 1 A line of Boehmite yAlOOH can also be used. 

Several procedures and modes of calculation can be used, e.g. standard 
additions or matrix suppression. The standard additions method is time 
consuming and requires the construction of curves of calibration. The 
technique of Chung (1974 a,b) with matrix suppression is faster and if 
required, makes it possible to identify the presence of amorphous 
minerals with short-range organization in the differential balance 4 . 
However it is necessary to obtain spectra whose principal reflections do 
not include diffuse bands or zones of superposition as the treatment of 
these signals is too complex. This method enables elimination of the 



4 If the soil sample contains undestroyed organic matter or substances that are 
transparent to X-ray, these substances can be found in the differential balance. 



X-ray Diffractometry 121 

matrix effects from the intensity - concentration, and all the intensities 
are obtained with only one scan, which reduces possible instrumental 
errors. 

Procedure 

A spectrum is performed on powder samples, on the pure corundum and 
on the sample mixture + corundum: 

- weigh a known weight of corundum (c) and a known weight of sample 
(A); the proportion of the mixture should be 1 : 1 

- homogenize in a horizontal mixer with a bowl and an agate ball for 20 
min 

- pour the powder into a support and pack slightly; level with a razor 
blade to obtain a smooth but not oriented surface; the reference plane 

must be perfect 5 . 

- place on a diffractometer under standard conditions using a Cu tube, a 
variable slit and a graphite monochromator with a time constant 
enabling accumulation of at least 20,000 counts per peak (minus 
background noise) and a 2 # scanning rate of 0.5° min -1 (or even 0.25° 
29 min -1 ); only scan the zone containing significant peaks 

- locate the position of the most intense diffracted peaks that are 
representative of each component, determine their intensity and 
compare with the internal standard - what does this dash imply? matrix 
suppressor. 

Calculations 

Based on the nature of monochromatic X-ray radiation of a defined 
wavelength, the nature of the matrix effects (absorption) and the basic 
equation of Klug and Alexander (1959), Chung mathematically extracted 
the effects of attenuation of mass: 

T kXlQ kXlQ 

XM t X t M, { } 

where 

I t is the intensity of the X-ray diffracted by a selected plane of component 

i (unknown); 



5 Certain authors prefer to prepare the samples by pelletization although there is 
a risk of causing a certain orientation of the powder due to the very strong 
pressure applied. However, there will be fewer errors connected with surface 
quality or the density of sites likely to diffract. 



122 Mineralogical Analysis 

k t is the constant which depends on the geometry of the diffractometer 

and the nature of component z; 
X t is the weight of the fraction of component z; 
Qt is the density of component i; 
jut is the coefficient of mass absorption (or attenuation coefficient of 

mass) of the pure component z; 
ju t is the coefficient of absorption of the total sample including component 

z, the internal standard and possibly a reference material. 
The last two terms characterize the effect of adsorption which is often 
difficult to measure with other methods. The introduction of a definite 
weight of a matrix suppressor (corundum resembling an internal 
standard) makes it possible to introduce: X f is the weight of the matrix 
suppressor (/, flushing agent) and X is the weight of the sample, and the 

equation: x f + X = X f + ^X. = 1 

i=\ 

n being the number of components of the sample, and 

(i,/rf)(W) = (W)(f,/f<f)> 

where I * and I i represent the intensities of the X-ray diffracted by a 

selected plane of each pure component. By introducing the ratio of 
intensity of reference K t = I/I c , and other substitutions, one arrives at the 
equation: 

X i = X f {kflk i )(l i II f ), (4.4) 

which gives the relation between intensity and concentration from which 
the effect of matrix is eliminated and which is used for quantitative multi- 
component analysis. With corundum as matrix suppressor the final simple 
equation (k f = k c = 1) is: 

*, = (*,/*,)(/,//.), (4.5) 

where 

Xi is the weight of the sample fraction; 

X c is the weight of corundum; 

It is the diffracted intensity of sample; 

I c is the diffracted intensity of corundum; 

k t = I/I c = intensity ratio of reference (Table 4.8). 



X-ray Diffractometry 123 

Remarks 

Using computerized equipment, it is possible to take into account the 
height of the reflection and the width at mid-height 6 or the surface of the 
d(001) reflections which the software calculates automatically taking 
stabilized background noise into account. 

Complementary measurements may be necessary on oriented samples: 
Mg 2+ saturated or solvated samples, and the use of multiplicative 
coefficients accounting for the structure of the minerals (fibrous clays 
with pseudo-layers that do not give very intense reflections, etc.). 

It is possible to combine the results of the average of two reflections as 
this can provide information on crystallinity, etc. The choice will depend 
on the shape of the diffractogram, the nature of the components, and the 
degree of precision desired. 

Table 4.8. Recommended values of /// c ratios (Eq. 5) from Bayliss (1986) 



Mineral 


I/Ic 


cf(A) 


Mineral 


I/I c 


cf(A) 


allophane 


0.1 


3.3 


illite- 
montmorillonite 


0.4 


12 


biotite 


9.0 


10.0 


kaolinite 1Md 


1.1 


7.1 


boehmite 


1.0 


6.11 


kaolinite 1T 


2.1 


7.1 


calcite 


3.7 


3.03 


muscovite 


2.2 


10 


corundum 


1.0 


2.09 


quartz 


4.3 


3.35 


dickite 


2.9 


7.2 


smectite 


3.0 


15 


gibbsite 


1.6 


4.85 


talc 


1.5 


9.3 


goethite 


1.4 


4.18 








gypsum 


2.2 


2.87 








hematite 


0.9 


2.70 








illite 


0.7 


10 









For the same mineral species, values of kj may vary with the geological 
origin and the nature of pedological alteration and measurements must 
must thus be carried out under the same conditions. The choice of minerals 
is made after chemical analysis and XRD. 



6 If it is necessary to compare samples by measuring intensity, it should be noted 
that the ratio of the heights of the peaks is only valid if the widths at mid - height 
are identical for the two samples. 



124 Mineralogical Analysis 

The Ij/I c ratio is affected by crystallinity, it approaches if the mineral is not 
crystalline (allophane - materials with short-range organization) and can 
reach 8-9 if the size of crystallites and crystallinity is optimum. Chemical 

2+ 2+ 

substitutions (e.g. heavy Fe minerals replacing light Mg minerals) cause 
variations in the Ij/I c ratio. 



4.3.3 Multi-Instrumental Quantitative Mineralogical Analysis 

Quantitative methods based on XRD have limited precision particularly 
when dealing with complex assemblies that give diffuse reflections or 
reflections that are more or less masked by superposition, or when there 
is a significant quantity of substances amorphous to X-rays. Multi- 
instrumental methods combine measurements based on XRD and other 
chemical and physical measurements making it possible to characterize 
the different elements. Measurements are generally made on clay 
fractions, but these measurements can be supplemented by others, for 
example organic matter destroyed by hydrogen peroxide, carbonates, soluble 
salts, iron oxides, etc. eliminated during extraction of the <2 |um clay 
fraction, and finally by analysis of sands and silts separated by sieving. 

These methods can only be used in specialized laboratories that have a 
wide range of instrumental methods such as XRD, IR, TEM, DTA-TGA, 
AA, ICP, etc., at their disposal. Each component of a mixture has its own 
chemical and physical characteristics that can be measured. First XRD 
spectra are qualitatively interpreted to identify clayey minerals (cf. Sect. 

4.2.4 and 4.2.5). 

Roberts (1974) and Robert et al. (1991) quantified the different 
elements using their specific properties. The organization, size, and shape 
of the particles make it possible the right choices and to enhance 
identification by methods like TEM-HR, STEM, EDX (see Chap. 8) of 
minerals that only present in small quantities and cannot be detected by 
XRD. Thermal analysis may be essential for the quantification of 
kaolinite, oxyhydroxides and chlorite. Losses between 110-300, 300- 
600, 600-950°C are used. Corrections for the oxidation of iron at high 
temperatures are required. 

Total chemical analysis of clay by a HF-HC1 attack (cf Chap. 31) 
makes it possible to determine the proportions of the different elements 
present, which is essential for the identification of the structure of the 
different mineral phases. Total K enables estimation of micas on the basis 
of 7.5% of K for illites compared to 8.3 - 10% of K for the other micas. 
Analysis by "selective" dissolution (cf. Chap. 6) using suitable 
procedures enables certain phases to be preferentially dissolved without 



X-ray Diffractometry 125 

the other elements in the matter undergoing a significant attack. For 
example, the attack of a sample (heated at 110°C for 4 h) by a 0.5 
mol (NaOH) L _1 solution followed by boiling for 2 min 30 makes it possible 
to extract allophane (42.7% Si0 2 -36.3% A1 2 3 ) and noncrystalline 
compounds such as colloidal silica (a little montmorillonite and 
vermiculite is dissolved). Fusion with pyrosulphate or possibly a tri-acid 
attack (H 2 S0 4 -HN0 3 -HC1) enables isolation of quartz and feldspars in 
the residue from the attack. This residue is then weighed. The weight 
should be increased by 3% to compensate for the slight dissolution of 
quartz and feldspars. The residue is then analyzed by XRD to detect the 
presence of feldspar then can be analyzed chemically after dissolution. 
Dissolution with Tamm reagent (in darkness or with UV photolysis) 
enables isolation of noncrystalline forms of iron and the CBD method 
(see Chap. 6) enables isolation of crystalline iron hydroxides. 

Analyses that identify the activity of clays can enable separation of 
certain types of 2:1 clay based on their cation exchange capacity using 
several different treatments: Na + saturation and displacement by Mg ++ , 
Mg 2+ saturation and displacement by ammonium acetate, etc. The total 
specific surface (external and/or internal) can be determined using the 
(EGME) method, by absorption of methylene blue, or by the BET 
method. 

Table 4.9. Some properties of minerals used for the adjustment of the results 





CEC 
(cmol kg" ) 


Specific surface 
(mV) 


Water loss 
540-900°C (%) 


mica 


25 


175 




0.50 


quartz 


2 


25 







feldspars 


2 


25 







allophane 


100 


800- 


1,000 





kaolinite 


5-10 


45 







colloidal Si02 


20 


200 




2 


montmorillonite 


80-100 


800 




0.88 


vermiculite 


175-200 


440 




1.83 


chlorite 


25 


175 




12.30 



All this quantitative information is imported into the software making it 
possible to specify the proportion of each component in the mixture, to 



126 Mineralogical Analysis 

assign the limits of the properties for the various compounds, and to 
select options for calculations. 



References 

Anton O and Rouxhet PG (1977) Note on the intercalation of kaolinite, dickitte 

and halloysite by dimethyl- sulfoxide. Clays Clay Minerals, 25, 259-263 
Bayliss P (1986) Quantitative analysis of sedimentary minerals by powder X- 

Ray diffraction. Powder diffraction, 1, 37-39 
Brindley GW and Brown G (1980) Crystal structure of clay minerals and their 

X-Ray identification. Miner alo. Soc., 415-438 
Calvert CS (1984) Simplified, complete CsCl-hydrazine-dimethylsulfoxide 

intercalation of kaolinite. Clays Clay Miner., 32, 125-130 
Chassin P (1974) Influence de la stereochimie des diols sur la formation des 

complexes interfoliaires de la montmorillonite calcique. Clay Miner., 11, 

23-30 
Chung FH (1974a) Quantitative interpretation of X-Ray diffraction pattern of 

mixtures. I- matrix flushing method for quantitative multi-component 

analysis. J. Appl. Crystallog., 1, 519-525 
Chung FH (1974b) Quantitative interpretation of X-Ray diffraction patterns of 

mixtures. II - Adiabatic principle of X-Ray diffraction analysis of mixtures. 

J. Appl. Crystallog., 7, 526-531 
Churchman GJ, Whitton JS, Claridge GGC and Theng BKG (1984) Intercalation 

method using formamide for differentiating halloysite from kaolinite. Clays 

Clay Miner., 32, 241-248 
Decarreau A (1990) Les poudres : techniques experimentales et interpretation 

des diagrammes - Facteurs determinant le mode d'empilement. In Structure, 

proprietes et applications. Societe Frangaise de Mineralogie et cristallographie, 

Groupe Fran^ais des Argiles, 209-236 
Eltantawy IM and Arnold PM (1974) Ethylene glycol sorption by homoionic 

montmorillonites. J. Soil Sci., 25, 99-1 10 
Gonzalez Garcia S. and Sanchez Camazano M. (1968) Differenciation of kaolinite 

from chlorite by treatment with dimethylsulfoxyde. Clay Miner. Bull., 1, 

447-450 
Klug HP and Alexander LE (1974) X-Ray diffraction procedures. Wiley 2nd edition 
Modre DZ and Dixon JB (1970) Glycerol vapor adsorption on clay minerals and 

montmorilllonite soil clays. Soil Sci. Soc. Am. Proc, 34, 816-822 
Olejnik S, Aylmore LAG, Posner AM and Quirk JP (1968) Infra-red spectra of 

kaolin mineral- dimethyl sulfoxide complexes. J. Phys. Chem., 72, 241-249 
Paterson E, Bunch JL and Duthie DML (1986) Preparation of randomly oriented 

samples for X.Ray diffractometry. Clay Miner., 21, 101-106 
Range KJ, Range A and Weiss A (1969) Fireclay type kaolinite or fire-clay 

mineral? Experimental classification of kaolinite - halloysite minerals. 

Proceedings of the International Clays Conference (Tokyo). Israel Universities 

Press, 3-13 



X-ray Diffractometry 127 

Roberts JM Jr (1974) X-Ray diffraction and chemical techniques for quantitative 

soil clay mineral analysis. Engineering Thesis, Pennsylvania State 

University, 78 pages 
Robert M, Hardy M and Elsass F (1991) Crystallochemistry, properties and 

organization of soil clays derived from major sedimentary rocks in France. 

Clay Miner., 26: 409-420 
Thomson A, Duthie DM, Wilson MT (1972) Randomly oriented powders for 

quantitative determination of clay minerals. Clay Miner., 9, 345-348 
Wada K and Yamada H (1968) Hydrazine intercalation, intercalation for 

differentiation of kaolin mineral, from chlorites. Am. Miner., 53, 334-339 



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Alekseeva TV, Alekseev AO, Sokolovska Z, Khainos M, Sokolowska Z and 
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Brindley GW and Brown E (1980) Crystal structure of clay minerals and their 
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Caillere S, Henin S and Rautureau M (1982) Mineralogie des argiles, 2, 
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Charley H (1989) Clay Sedimentology., Springer Belin Heidelberg New York, 623 p 

Dixon JB and Weed SB (1989) Minerals in soil environments., Soil Science 
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Gautheyrou J and Gautheyrou M (1979) Etude des argiles par diffraction X. 
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128 Mineralogical Analysis 

Kovda IV, Morgun EG, Tessier D, Pernes M (2000) Particle orientation in 

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Chen PY (1977) Table of key lines in X-Ray powder diffraction patterns of 

minerals in clays and associated rocks. Dept. of Natural resources, (Indiana, 

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Robert M (1975) Principes de determination quantitative des mineraux argileux 

a l'aide des rayons X. Problemes particuliers poses pour les mineraux 

argileux les plus frequents dans les sols des regions temperees. Ann. Agron., 

26, 363-399 
Stucki JW (Goodman BA and Schwertmann U (1985) Iron in soils and clay 

minerals., D. Reidel, 893 p. 
Teissier D (1984) Etude experimental de V organisation des materiaux argileux. 

Hydratation, gonflement et structuration au cours de la dessiccation et de la 

rehumectation., INRA, These doc. Etat, 361 pp. 
Thorez (1975) Phyllosilicates and clay minerals. A laboratory handbook for 

their X-Ray diffraction analysis., Lelotte Ed., 579 p 
Wilson MJ (1987) A handbook of determinative methods in clay mineralogy., 

Blackie - Chapman and Hall, 308 p. 



Preparation of Oriented Aggregates on Porous Ceramic Plate 

Kinter EB and Diamond S (1956) A new method for preparation and treatment 

of oriented- aggregat specimens of soil clays for X-Ray diffraction analysis. 

Soil ScL, 81, 111-120 
La Manna JM and Bowers FH (1985) A suction apparatus for orienting clay 

minerals into porous ceramic tile. Soil Sci. Soc. Am. J., 49, 1318-1319 
Rich CI (1969) Suction apparatus for mounting clay specimens on ceramic tile 

for X-Ray diffraction. Soil Sci. Soc. Am. Proc, 33, 815-816 
Shaw HF (1972) The preparation of oriented clay mineral specimens for X-Ray 

diffraction analysis by a suction unto ceramic tile method. Clay Miner., 9, 

349-350 



X-ray Diffractometry 129 

Saturation of Clays by Cations 

Brindley GW and Ertem G (1971) Preparation and solvation properties of some 

variable charge montmorillonite. Clays Clay Miner., 19, 399-404 
Buhmann C, Fey MV and De Villiers JM (1985) Aspects of the X-Ray 

identification of swelling clay minerals in soils and sediments. S. Afri. J. 

ScL, 81, 505-509 
Calvet R and Prost R (1971) Cation migration into empty octahedral sites and 

surface properties of clays. Clays Clay Miner., 19, 175-186 
Hofmann U and Klemen R (1980) Verlust der Austanschfahigeit von 

lithiumionen an bentonit darch erhitzung (perte d'echangeabilite des ions 

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95-99 
Lim CH and Jackson ML (1986) Expandable phyllosilicate reactions with 

lithium on heating. Clays Clay Miner., 34, 346-352 



Saturation, Solvation, Intercalation Complexe, Dissolution 

Barnhisel RI and Bertsch PM (1989) Chlorites and hydroxy- interlay ered 

vermiculite and smectite. In Minerals in soil environments Dixon JB and 

Weed SB ed., Soil Sci. Soc. of Am., 729-740 
Barnhisel RI (1977) Chlorites and hydroxy interlay ered vermiculite and 

smectite, 331-356. In Minerals in soil environments, Dixon JB and Weed SB 

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Brindley GW (1966) Ethylene glycol and glycerol complexes of smectites and 

vermiculites. Clay Miner., 6, 237-260 
Brindley GW and Ertem G (1971) Preparation and solvation properties of some 

variable charge montmorillonites. Clays clay Miner., 19, 399-404 
Churchman GJ (1990) Relevance of different intercalation tests for 

distinguishing halloysite from kaolinite in soils. Clays and clay Minerals, 

38, 591-599 
Follet EAC, McHardy WJ, Mitchell BD and Smith BFL (1965) Chemical 

dissolution techniques in the study of clays. Part 1. Clay Miner., 6, 23-24 
Novich BE and Martin RT (1983) Solvation methods for expandable layers. 

Clays Clay Miner., 31, 235-238 
Suquet H, Iiyama JT, Kodama H and Pezerat N (1977) Synthesis and swelling 

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242 
Suquet M, Calle de la C and Pezerat H (1975) Swelling and structural 

organization of saponite. Clays Clay Miner., 23, 1-9 
Theng BKG, Churchman GJ, Whitton JS and Claridge CGC (1984) Comparison 

of intercalation methods for differentiating halloysite from kaolinite. Clays 

Clay miner., 32, 249-258 
Walker GF (1958) Reactions of expanding-lattice clay minerals with glycerol 

and ethylene glycol. Clay Miner. Bull., 302-313 



130 Mineralogical Analysis 

White JL and Jackson ML (1947) Glycerol solvation of soil clays for X-Ray 
diffraction analysis. Soil Sci. Soc. Am. Proc, 11, 150-154 



Preparation of Iron Oxides 

Brown G and Wood IG (1985) Estimation of iron oxides in soil clays by profile 

refinement combined with differential X-Ray diffraction. Clay Minerals, 20, 

15-27 
Campbell AS and Schwertmann U (1985) Evaluation of selected dissolution 

extractants in soil chemistry and mineralogy by differential X-Ray 

diffraction. Clay Miner., 20, 515-519 
Meunier A and Velde B (1982) X-Ray difffraction of oriented clays in small 

quantities (0,1 mg). Clay Miner., 17, 259-262 
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samples for X-Ray diffractometry. Clay Miner., 21, 101-106 
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Schwertmann U, Murad E and Schulze DG (1982) Is there Holocene reddening 

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223 
Torrent J, Schwertmann U and Schulze DG (1980) Iron oxyde mineralogy of 

some soils of two river terrace sequences in Spain. Geoderma, 23, 191-208 



Quantitative XRD 

Austin GS and Leininger RK (1976) Effect of heat-treating mixed-layer illite- 

smectite as related to quantitative clay mineral determinations. J. Sedim. 

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mineral mixtures. Miner. Mag., 49, 531-538 
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Cody RD and Thomson GL (1976) Quantitative X-ray powder diffraction 

analysis of clays using an oriented internal standard and pressed discs of 

bulk shale. Clays Clay Miners, 24, 224-231 
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as a function of quantitative estimations by X-ray diffraction. Bull. Soc. 

Beige Geol, 94, 275-281 
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calcite by X-ray diffraction effect of grinding on peak height and peak area. 

Sediment, 20, 437-444 



X-ray Diffractometry 131 

Goehner RP (1982) X-Ray diffraction quantitative analysis using intensity ratios 

and external- standards. Adv. in X-Ray Analy., 25, 309-313 
Heath GR and Pissas NG (1979) A method for the quantitative estimation of 

clay minerals in North Pacific deep sea sediments. Clays Clay Miner., 27, 

175-184 
Hogson M. and Dudgney ANL (1984) Estimation of clay proportions in 

mixtures by X-Ray. Diffraction and computerized chemical mass balance. 

Clays and Clay Miner., 32, 19-28 
Hooton DH and Giorgetta NE (1977) Quantitative X-Ray diffraction analysis by 

a direct calculation method. X-Ray Spectrom., 6, 2-5 
Hubbard CR and Smith DK (1977) Experimental and calculated standards for 

quantitative analysis by powder diffraction. Adv. X-Ray Anal., 20, 27-39 
Hubbard CR, Evans EH and Smith DK (1976) The reference intensity ratio II I c 

for computer simulated powder patterns. J. Appl. Cry St., 9, 169-174 
Johnson LJ, Chu CH and Hussey GA (1985) Quantitative clay mineral analysis 

simultaneous linear equations. Clays and Clay Miner., 33, 107-1 17. 
Kahle M, Kleber M and Reinhold J (2002^ Review of XRD-based quantitative 

analyses of clay minerals in soils: the suitability of mineral intensity factors. 

Geoderma, 109, 191-205 
Norrish K and Taylor RM (1962) Quantitative analysis by X-Ray diffraction. 

Clay Miner. Bull, 5: 98-109 
Ouhadi VR and Yong RN (2003) Impact of clay microstructure and mass 

absorption coefficient on the quantitative mineral identification by XRD 

analysis. .4/?/?/. Clay Sci., 23, 141-148 
Parrot JF, Verdoni PA and Delaune-Mayere (1985) Analyse modale semi- 
quantitative d'apres l'etude des Rayons X. Analusis, 13, 373-378 
Pawloski GA (1985) Quantitative determination of mineral content of geological 

samples by X-Ray diffraction. Am. Mineral, 70, 663-667 
Persoz (1969) Fidelite de l'analyse quantitative des poudres de roches par 

diffration X. Bull. Centre Rech. Pau (SNPA), 3, 324-331 
Renault J (1987) Quantitative phase analysis by linear regression of chemistry 

on X-Ray diffraction intensity. Powder Diffract., 2, 96-98 
Ruffell A. and Wiltshire P. (2004) Conjunctive use of quantitative and 

qualitative X-ray diffraction analysis of soils and rocks for forensic analysis. 

Forensic Science International 145, 13-23 
Taylor RM and Norrish K (1972) The measurement of orientation distribution 

and its application to quantitative determination of clay minerals. Clay 

Miner., 9, 345-348 
Tomita K and Takahashi H (1985) Curves for the quantification of mica/smectite 
and chlorite/smectite interstratifications by X-Ray powder diffraction. 
Clays Clay Miner., 33, 379-390. 



Mineralogical Analysis by Infra-Red 
Spectrometry 



5.1 Introduction 



5.1.1 Principle 

The interaction of matter with infra-red (IR) radiation makes it possible to 
characterize energies of vibration of the molecules on several components 
(Fig. 5.1): along the axis of the chemical bonds (vibrations of valence or 
stretching, which, apart from diatomic molecules, are seldom pure) and 
deformations that are perpendicular to the bond axis (rotation, torsion, 
shearing, swinging, librations, bending). IR radiations correspond to these 
energy levels. IR absorption occurs when the frequency of the radiation is 
equal to that of the vibrations. 



Axis 



A 



•vwiOvw* 



A A A-A A 



Stretching Translation Bending Waging Rocking Twisting 

Fig. 5.1. Examples of vibration of a simple polyatomic molecule 



134 Mineralogical Analysis 

IR adsorption spectroscopy uses radiations ranging between the visible 
waves and microwaves. This field is usually divided into three energy 
zones: near-IR, medium IR and far IR (Fig. 5.2). In these zones, different 
molecular vibrations correspond to the energy of IR radiations. 

- Near infra-red (NIR), as well as visible and UV, account for the high- 
energy electronic spectra related to fundamental orbitals, for example 
the change from a link orbital to an empty orbital of higher energy; NIR 
spectrometry is currently being developed for the study of soil organic 
matter (cf. Sect. 5.3.1 and Part 2 of this book). 

-Medium IR, ranging between 300 and 5,000 cm" 1 makes it possible to 
observe vibrations involving protons, (vibrations that correlate well 
with structure and whose transitions correspond to slight modifications 
in stretching or deformation of the bond angles in the molecule). Unit 
cells of clays (crystallographic units) contain polyatomic ions or 
molecules whose internal modes of vibrations occur between 4,000 and 
approximately 400 cm" 1 . These vibratory states have been the subject of 
detailed studies in mineralogy. Absorption bands make it possible to 
characterize active molecular groupings satisfactorily. 

- Other modes of vibrations which come from the lattice can occur after 
displacement of a polyatomic group within a unit cell in far IR at very 
low frequencies between 200 and 10 cm 1 . This field, which has not 
been extensively explored to date, is now accessible thanks to the 
development of IR spectrometers. Bands of rotational transitions that 
are not widely spaced enable quantification of the number of 
revolutions around an axis without stretching or notable modification of 
the bound angles that are characteristic of the geometry of the crystal. 

IR spectrometry is thus used as a complement for X-ray diffraction and 
chemical and thermal analysis. XRD (cf. Chap. 4) expresses long- 
distance periodicity satisfactorily, but is not effective in the case of 
substances that are amorphous to X-ray or minerals with short-range 
arrangement that appear during sequences of alteration. 

With IR, a spectral signature can be identified, along with the nature 
and the direction of the bonds, and our understanding improved of atomic 
structures, as well as of the degree of isomorphic substitution in the 
tetrahedral (Si-Al) and octahedral (Al-Mg) layers. These data are needed 
to identify certain minerals, to quantify molecular water and constitutive 
hydroxyls and to detect the presence of crystalline or non-crystalline 
impurities, which influence the regularity of the lattice structure. 

In clays and clay minerals only some molecular groups are likely to 
vibrate and the spectra are often less complex than for certain organic 
substances. 



IR Spectrometry 



135 



Visible 



Near IR 



Medium Infra-Red 



FarlR 



Microwaves 



3.5 4 4.5 5 5.5 6 



8 9 10 12 16 20 



3 000 

I 



2 000 1 600 
I I I 



3 100 
' ' i 



X |Lim 

-1 
n cm 



n cm 



Quartz 



LiF 



CaF 9 



NaCI 



KBr PE 



BaF 9 



MULL 



Fluorolube 



Electronic 
Transition 



Valence strength field - functional groups 

Stretching vibration transitions 



IRTRAN 



AgCI |Cs 



NUJOL 



Ranges of 
optical 

transparency 
of supports 



Spectral signature 



- Molecular 
deformation 
Angular modifications - libration 



Spectral 
range 



Fig. 5.2. Field of infra-red molecular spectrometry and transparency of the 
optical elements. The position of the bands in x-coordinate is expressed 
either in wavelength X in nm or |im, or often in wavenumber 
J 1° In ordinate, transmittance expresses percent 



v{cm) 

A (cm) A(jum) 

of radiation that crossed the sample (PE 



polyethylene, Mylar) 



5.1.2 IR Instrumentation 



A spectrometer includes an IR source, optical elements, a detector and a 
rack of computerized measurements. It is difficult to choose optical 
equipment that covers the whole spectrum from near IR to far IR 
(Fig. 5.2). 

The source must emit intense polychromatic radiation covering the 
whole of the IR spectrum. A filter eliminates UV and visible radiations 
that are emitted simultaneously. 

- Sources with filaments of tungsten cover only the field of near IR; 

- nickel-chromium filaments make it possible to reach 600 cm" 1 ; 

- mercury vapour lamps make it possible to reach far IR between 300 and 
10 cm" 1 ; 

- globars (carborundum rods with refractory oxides), which are often 
used with water cooling to stabilize the temperature of the source at 
around 1,500°C, emit up to approximately 200 cm" 1 ; 

- silicon carbide sources emit between approximately 6,500 and 50 cm" 1 . 

Optical equipment (e.g. lenses, windows, dispersive systems) should 
be selected for their transmittance properties (Fig. 5.2) and resistance to 
water or solvents. The lenses should preferably be replaced by mirrors 
(Figs. 5.3 and 5.8). 

Filtering of the visible spectrum is generally accomplished by surface 
treatments with germanium, or black polyethylene films of varying 
thickness. 

The choice of the detector is also limiting and its surface area, the 
spectral field covered, the sensitivity and the response time of the 
apparatus, the frequencies to be detected, maintenance needs (operation 



136 Mineralogical Analysis 

under liquid nitrogen or helium) should all be taken into account. The 
detector can be: 

-non-selective e.g. a thermocouple or thermopile (several couples 
connected in series), a bolometer with doped germanium for far IR that 
operates in liquid helium (2 K), MCT detectors (Mercury, Cadmium, 
Tellurium), DTGS detectors (Deuterium enriched Triglycine Sulphates) 
thermostated for medium IR, detectors of Golay with a gas chamber for 
far IR; these detectors are very sensitive, but very fragile; 
- selective (photon-electron transformation as a function of the 
wavelength), e.g. lead sulphide, lead selenide, or indium antimonide 
detectors that function in liquid nitrogen. 

The optical system can be based on a dispersive mode or managed by 
interferometry in a mono or double beam system. 

In dispersive mode radiation crosses the sample from where it partially 
arises (transmission-absorption) to strike a dispersive lattice or a 
monochromator which divides the beam as a function of wavelength 
(Fig. 5.3a). Energy is recorded point by point by rotation of the lattice. It 
is first necessary to determine the zero point of the instrument, and then 
to determine the basic spectrum without a sample to take into account in 
particular the C0 2 and H 2 in the air. Transmittance is calculated at each 
wavelength by the ratio of the two signal-to-noise values. The resolution 
is often insufficient and energy decreases with an increase in wavelength, 
which makes it necessary to gradually open the slit of the monochromator 
and modify the background noise. These operations can be automated, 
and the degree of precision can be increased by the use of a double beam 
and measurements on sample and blank taken alternately at each 
wavelength with the help of a modulator. In this way the energy of the 
beam is maintained constant. The study of far IR is not possible because 
of the nature of the apparatuses and insufficient energy. Ratiometric 
apparatuses are slow and top-of-the-range models are very expensive, as 
several high resolution lattices are necessary for satisfactory linear 
dispersion. 

Better performances can be obtained by using an interferometric 
system linked with data processing by Fourier transform (Fig. 5.3b). 
Interferometry is based on rapid movements of a mirror. Each wavelength 
is modulated at a characteristic frequency determined by the speed of the 
mirror. The recording of the complex data gives an interferogram that is 
treated in real time by the Fourier transform. This makes it possible to 
obtain a spectrum where the amplitude of the signal is recorded as a 
function of the frequency. 



IR Spectrometry 



137 



Blank 



I R source 



O^— 0- 



a Sample 

Ix 



,W 



ZD-m 




I R source 



w 




7 




6 




5 




Detector J 



Fig. 5.3. Types of Infra-red spectrometers: (a) Dispersive 1: dispersive system: 
prisms, lattices, monochromator, continuous high resolution interferential 
wedge, 2: exit slit for precise selection of the field of frequency, 3, 4, 5: 
detection, amplification and acquisition, / /, l x '■ total incidental and 
transmitted intensities at the selected wavelength x, T: transmittance /// 
corresponding to the absorbance A = - log 7(b) With interferometer and 
Fourier transform 1: separation made of KBr with germanium film (400- 
4,000 cm" 1 ) or made of MYLAR (0-700 cm" 1 ); 2: mobile mirror, spectral 
composition is a function of the position of the mirror, function resolution 
of the amplitude of displacement, 3: He-Ne laser for measuring 
movements of the mobile mirror and indexing mirror direction, 4: sample 
compartment under vacuum or controlled atmosphere, 5: 
intensity/position of mirror, 6: interferogram, 7, 8: real-time data 
acquisition system 



In the Michelson interferometer, the polychromatic radiation of the 
source is divided into two beams by a separation made of KBr or Mylar 
(covered with a germanium film to filter radiations of the visible 
spectrum). One of the two beams is sent to a fixed mirror, the other to a 
mobile mirror that is moved at a known speed by to a linear motor. The 
two beams are than recombined by the beam splitter. The resolution 
depends on the maximum stroke of the mobile mirror. Alignment must be 
maintained during the movement of the mobile mirror with no vibrations 
or slope likely to deform the spectra and to give erroneous transmittance 
values. An in-out movement of the mirror allows simultaneous analysis 
of all the wavelengths of IR radiation. 

The changes in the signal reflect variations in modulation of the 
interferogram. The results are recorded at the different positions of the 
mirror, which requires knowing its exact position by laser radiation. Each 
individual signal is located by multiplexing and decoding. The whole 
spectrum is scanned sequentially as a function of time in less than one 
second. 



138 Mineralogical Analysis 

To limit the influence of moisture and atmospheric CO2, the optical 
circuits function in a sealed enclosure, except for the sample 
compartment which can be purged with dry nitrogen. 

The resolution is generally about 2-4 cm" 1 but can reach 0.1 cm" 1 at 
the price of additional memory which otherwise requires a powerful 
information processing system. 

FTIR apparatuses are generally mono-beam and more rarely, in top-of- 
the-range models, double-beam which enables the signal-to-noise ratio to 
be maintained uninterrupted. Suitable software makes it possible to 
control the spectrometer, collect the results and process the data in real 
time to restore the spectrum. If necessary, a database search makes it 
possible to compare the spectra with IR bases for identification, 
calibration, subtraction of spectra, etc. Calibration is carried out with 
polystyrene blades that have narrow intense bands distributed between 
699 and 3104 cm" 1 . 

In practice, the choice of an apparatus is based primarily on the 
scientific objectives which determine the main requirements: the 
wavelength field to be scanned, the nature of materials to be analysed 
(solid, liquid, gas), the required sensitivity, resolution, data-processing 
capacity and quality of the software, possible extensions of the basic 
module (coupled with e.g. liquid or gas chromatography, EGA thermal 
analysis, Raman spectroscopy, IR or Raman microscopy). As far as price 
is concerned, instead of purchasing a very expensive top-of-the-range 
"universal" apparatus, two dedicated complementary apparatuses can be 
adapted for repetitive laboratory tasks. 

For routine soil analysis, an apparatus that covers medium IR up to 
220-250 cm" 1 with an optical system in KBr or better still in Csl and a 
resolution of about 2 cm" 1 is sufficient, but for a more specialized 
laboratory, access to the field of far IR is now necessary. 



5.2 IR spectrometry in Mineralogy 

5.2.1 Equipment and Products 

- Ultra-microbalance, 10" 6 g sensitivity, range 2-5 g 

- IR or FTIR spectrometer suitable for the required ranges of wave- 
lengths 



I R Spectrometry 139 

- IR microscope 

- binocular magnifier (x 100) 

- pellet press (10 tons cm" 2 ) 

- centrifuge with centrifugation chambers (cf. Chap. 4) 

- cooled moisture-proof vibrating grinder with agate ball for crushing in 
liquid medium 

- drying oven (105°C) with precise electronic regulation 

- Pyrex glass desiccator 

- lab glassware 

- quality IR products: KBr, Csl, polyethylene, Nujol (paraffin oil), AgCl, 
CaF 2 , IR TRAN 4, solvents, hexachlorobutadiene (Voltalef oil), 
Fluorolube (fluorinated hydrocarbon oil), various mineral standards. 



5.2.2 Preparation of the Samples 

Types of Preparation 

The preparation of the sample for analysis by IR spectrometry is of prime 
importance: it conditions the spectral field of analysis and its limits, and 
indirectly, the sensitivity and the selectivity of measurements. 

Determination must be performed on elementary particles that do not 
exceed 5 |um and even 1 or 2 |um if the MULL technique is used or if 
quantitative analyses are required. 

Clay fractions with a particle size <2 |um in H + , NH 4 + or X + form, or 
better fractions <0.5 |um purified with a standard Sharpies ultra-centrifuge 
(cf. Chap. 3) are usually used for analysis. Because of the size of their 
unit cell, clays display only one negligible distortion of the absorption 
bands. Crush the samples and homogenize with the agate mortar in the 
presence of a volatile organic liquid that is inert to IR (Ethanol, acetone, 
etc.); avoid modifying or destroying the crystalline structure. Too large 
particles could cause spectral distortion, dispersion of incidental radiation 
and widening of the absorption bands (Christiansen effect). 

Destruction of the organic matter is often necessary first to limit 
organic absorbance, which is likely to overload the spectrum, and second 
to avoid excessive retention of adsorbed water, which can mask some 
absorbance of minerals. However, in some cases this treatment can lead 
to neogenesis of minerals. 



140 Mineralogical Analysis 

For measurements on solid samples 

- In transmission-absorption, clays can be analysed in three main forms: 

a. thin films that are self-supporting or placed on suitable supports, 

b. discs made with binding agents that are transparent to IR, 

c. in a mixture with liquid mulls. 

- In MR or Raman diffuse reflectance, the sample is simply packed in a 
sample holder taking care to limit preferential orientations. 

- In multiple specular reflectance or attenuated total reflectance (ATR), 
the problems of interface with the soils are often not reproducible and 
these techniques can generally only be used on thin blades. 

- In IR microscopy samples can be analysed on film or as microsamples 
without preparation. 

Pretreatments can cause changes in the properties of the samples: these 
can be used in comparisons with chemically untreated rough samples 
preserved in their original conditions (amorphous phases, etc.). 

For measurements on liquid samples separated from the soil by 
chemical means, it is possible to perform the measurements either (i) in 
solvents that are transparent to IR and do not react with the minerals, or 
(ii) NIRS measurements on freeze-dried extracts. 

For measurements on gas samples resulting from controlled pyrolysis 
or decomposition of mineral fractions during thermal analyses (EGA and 
TGA-DTA, see Chap. 7), sample "powders" are used that are identical to 
those used for standard thermal analyses. 

To improve sensitivity, special cells are used to lengthen the path of 
the beam in the gaseous medium, absorption being proportional to length. 
Preliminary purging of the air in the sample compartment is required to 
eliminate any H 2 and C0 2 present. 

Preparation of Self-Supporting Films 

Technique 

This preparation is only possible with certain types of clay, e.g. 
montmorillonites, vermiculites and fibrous clays, that can be prepared as 
thin but stable films. 

The method of Farmer and Palmieri (1975) has been slightly modified 
to allow quantification of the measurements. This technique has the 
advantage of not subjecting the sample to a strong pressure and of 
avoiding exchange reactions between the sample and the binding agent 
added in the pelletizing method. The main difficulties are the critical 
thickness of the film (maximum 4-8 |um), its capacity to transmit a 
sufficient intensity, its mechanical resistance and the frequent difficultly 



IR Spectrometry 



141 



involved in its removal, which requires great technical skill. Silicated 
minerals often deposit with preferential orientation, which makes it 
possible to identify the vibrations caused by the oscillation of the dipoles 
which should be perpendicular to the plan of the lattice. It is also possible 
to study polychroism related to the plane using a goniometer. 



\ 



Clay 
suspension 



Chamber 
4ml_ 




Sealing 
device 

Polythene 
film 

Slide support 




Fig. 5.4. Preparation of self-supporting mineral films: (a) centrifugation at 2,500 g 
in Cyto Hettich chamber, (b) removal of self-supporting film 



Procedure 

- Crush a clayey extract of known weight with an agate mortar with a 
little water to produce a fluid paste, and then put in suspension in a 
known volume of distilled water 

-transfer the complete suspension on a slide covered with a flexible 
polythene film in a Cyto Hettich chamber (Fig. 5.4); centrifuge at 
moderate speed (approximately 2,500 g) 

the selected volume of the chamber is 4 mL, making it possible to 
obtain a film 12.4 mm in diameter, that is to say a surface area of 120 
mm 2 ; in this way it is possible to determine the quantity deposited per 
unit area (the density must be approximately 1-2 mg per cm 2 ) 

- remove the clear supernatant solution with a pipette and air dry the film 
containing the deposit 

- remove the film by passing the flexible support across the edge of a 
bevel-edged blade and transfer it onto a support for IR measurement; 
the deposit can also be transferred onto a thin filter support with rigid 



142 Mineralogical Analysis 

polymeric mesh, which avoids migration thanks to its continuous 
structure 
- store the deposit for 48 h in a desiccator on P 2 5 before analysis. 

Preparation of Film on a Support Transparent to IR 

Principle 

This system makes it possible to obtain thin films either for oriented 

deposit by simple gravity or by centrifugation as above. 

The following factors have to be taken into account: the thickness of 
the clay deposit, the choice of a suitable solvent to avoid dissolution of 
the support, the spectral field useable with this type of support, the 
reactivity of the clay-solvent-support. In general, KBr slides are used 
because they are cheap and easy to use, and enable medium-IR scanning 
up to approximately 400 cm" 1 , or Csl up to 220 cm" 1 . Polythene is used 
for far IR. The sample is put in suspension in a solvent without 
dissolution. The surface can be impregnated by microvaporization with 
Nujol to limit reflectance phenomena at the air-clay interface. 

Procedure 

Depending on the IR domain, select a pellet 13 mm thick obtained by 
pressure (10 tons cm" 2 AgCl, CaF 2 IRTRAN 2, IRTRAN 4, Ge, Si, KBr, 
Csl, polythene, etc.). 

The sample in suspension in a suitable organic solvent is deposited in 
the same way as in the preceding procedure by gravity or centrifugation. 
After drying, carefully heat the disc covered with film in a drying oven at 
100°C for 5 h to eliminate all traces of water, then store in a desiccator 
until analysis. 

Preparation of Discs (Solid Solution) 

Principle 

The solid sample is completely pulverized with an agate mortar in the 
presence of an organic liquid (for example ethanol) then dried under 
vacuum in the desiccator with phosphoric anhydride. It is then mixed 
with a matrix that enables to form self-sustaining discs at high pressure; 
these disks can be used with quite a wide range of IR radiation. 

For swelling smectites, it is preferable to grind a known weight of 
sample with a little water to form a thick paste, then add the binding 
agent and grind the wet sample again. After complete drying, 
homogenize the diluted sample with a microball grinder. 

This system is easy to use and is the most widely used, but it has 
certain disadvantages: 



IR Spectrometry 143 

- all materials used as binding agents for the discs have limited 
transmission in IR so it is impossible to observe the absorbance of a 
clay on the whole range from near IR to far IR on only one pellet 

- the reactivity of the support may result in exchange reactions with clay. 

For example potassium bromide (KBr), which is transparent up to 400 
cm" 1 (Fig. 5.2), produces excellent pellets that allow good spectra in a 
wide range of IR. But with 2:1 clays, such as smectites which contract 
with K + , exchange phenomena can cause deformation of the absorption 
spectra (Nyquist and Kagel, 1971). These discs are thus not suitable for 
studies concerning adsorbed water as K reduces water retention, or for 
the study of surface cations. 

As KBr is hygroscopic, the pellets have to be stored in a desiccator 
under vacuum with phosphoric anhydride to limit the phenomena of 
adsorption of water and resulting uncertainties in interpretation between 
the bands of hydroxyls and those of water adsorbed by minerals and the 
support. For far IR, polyethylene, polytetrafluoroethylene (Teflon), or 
paraffin should be used. 

Pressure can cause inappropriate transformations by amplifying the 
effect of the chemical reactivity, but can be used for in situ study of the 
effects of very high pressures obtained with of a diamond-cell anvil 
making it possible to analyse by IR the induced transformations (Weir 
et al., 1959; Liu and Mernagh, 1992). 

Procedure (Qualitative Analysis) 

- Choose the matrix and the diameter of the pellets 

- dry the sample in a desiccator for 48 h to eliminate non-structural water 
whose 3,440 cm" 1 band can mask that of structural OH; this method of 
drying is not suitable for all minerals (cf. Chap. 1) 

~ dry the finely crushed IR-quality binding agent in the drying oven under 
vacuum at 100°C for one night if its thermal stability permits 

- for a disc of approximately 1 mm thickness and a diameter of 13 mm, 
weigh 0.5-3 mg of clay sample at 0.01 mg precision. 

- add 300 mg of KBr (can be changed) 

- homogenize with a microhomogenizer with a plastic or agate ball for 2 
min, or grind with the agate mortar once more for a perfect mix 

- transfer in a stainless steel mould 13 mm in diameter (A in Fig. 5.5b). 

- apply light pressure and degas under vacuum for 5 min 



144 



Mineralogical Analysis 



10 tons 




b 


I 


A 




A 




C 






I \ 


^ D 


Sample - 




I I 








Nil 




B 





Vacuum 




Fig. 5.5. Preparation of the samples in solid solution (a):12 ton manual hydraulic 
press, (b): detail of a pelletizer: A body, B: removable base, C 13 mm § 
plunger, D: 13mm 4> polished cylinders, E: release ring from the mould, 
filled circle represents sealing ring. 

- Press (Fig. 5.5a) at 10 tons cm" 2 for 10 minutes (KBr becomes plastic at 
this pressure) 

- extract the disk from the mould using the release ring (E in Fig. 5.5); it 
should look homogeneous, smooth and transparent; do not touch it with 
the hands 

-desiccate at 100°C for 2 h and store in a desiccator on P 2 until 

needed. 

In practice, it is advantageous to make two of even three pellets using 
the same binding agent at two different concentrations: 

- a pellet made with 3 mg of sample for 300 mg of KBr to reach total 
absorption in the zones around 1,000 cm" 1 and 500 cm" 1 (silicates) 

-a pellet made with 0.25-0.5 mg of clay sample to obtain details of the 
spectrum in the areas of intense absorption of silicates 

- a third pellet made with a binding agent transparent to far IR will also 
be needed if spectra below 200 cm" 1 are required 

- perform the spectra under the instrumental conditions selected. 



The time of passage in the spectrometer will depend on the type of 
material (dispersive or interferometer), the scanning zone with dispersive 
apparatuses, or the resolution required. After measurement, the pellets 



can be stored in the desiccator on PO . 



IR Spectrometry 145 

To avoid corrosion, the pelletizer must be cleaned immediately after 
use without abrasion. 

Preparation of the Clay Samples in the Form of Mull 

Principle 

When interactions are possible between the binding agent and the sample, 
when it is impossible to make self-supporting films or when pressure is 
not desirable, the Mull technique can be used with a non- volatile inert oil. 
Nujol (paraffin oil), hexachlorobutadiene (Voltalef oil) or Fluorolube 
(fluorinated hydrocarbon oil) is mixed with the sample to form a fluid 
paste which is pressed between two windows. The method is rapid but 
only qualitative; the sample is oriented. It is not possible to desiccate the 
mixture in the drying oven. 

Procedure 

-Place 10 mg of dried clay sample in a 50 mm agate mortar; add a 

known quantity of Mull to moisten the powder using a micropipette or 

a spatula 

- crush to obtain a thick paste in which the sample is uniformly dispersed 
(concentration will be about 0.3-0.5%) 

- spread out the mixture with a spatula on a window transparent to IR, 
then cover with another window to obtain a regular thickness taking 
care not to trap air inside 

- place in the spectrometer and record the spectrum. 

The spectral limits of the windows and the zones of absorption specific 
to the Mull matrix can be taken into account in interpretation. For 
example, Nujol strongly absorbs between 3,000 and 2,800 cm" 1 (CH), 
1,460 cm" 1 , 1,375 cm" 1 , Fluorolube does not absorb between 4,000 and 
1,400 cm" 1 . Both mulls are complementary and two mulls should be 
made to check that the absorption bands do not mask the bands specific to 
the sample. The homogeneity of the suspension is difficult to obtain and 
maintain: prepared samples should thus always be stored horizontally. 

Preparation for Specular or Diffuse Reflectance, Attenuated 
Total Reflectance (ATR) 

As the intensity of absorption depends on the angle of incidence, the 
surface should present weak granulation, and isogranulometric grinding 
to 0.2 mm is thus necessary. Compression should be carried out as for 
XRD powders (cf. Chap. 4) avoiding excessive orientation. 



146 Mineralogical Analysis 

The thickness of the powder (approximately 1 mm) is not critical, since 
the radiation only penetrates a few microns. In certain cases, it is possible 
to use compressed pellets with or without the addition of binding agents, 
but in this case the orientation is rather strong. It should be noted that 
since the refraction index is significant in measurements by reflectance, 
significant differences will be observed between the spectra obtained by 
transmission and by reflectance at high wavelengths. 

Deuterization 

In specialized laboratories, deuterization is an ideal method to study 
water in clays. In heavy water, deuterium replaces hydrogen. When H 2 
is replaced by D 2 0, the OH of the interstitial water is deuterated, but not 
the OH of the lattice (Wada, 1966). The interatomic distances do not 
change, the mass is doubled and the vibration frequencies drop. It is thus 
possible to separate the reticular OH or adsorbed water, and eliminate the 
ambiguity of the measurements in studies of mineral gels (Nail et al., 
1976). 

Remarks 

During preparation, atmospheric contamination of the rough samples 
must be avoided, for example: 

-ammonium can produce absorption around 3,250 and 1,400 cm" 1 
(stretching and deformation) 

- in the presence of calcium, attacking organic matter with hydrogen 
peroxide can lead to the formation of insoluble calcium oxalate which 
produces absorption near 1,400 cm 1 , the destruction of organic matter 
with sodium hypochlorite does not give oxalate and is consequently 
more suitable in this particular case 

- note that oxidation of organic matter is accompanied by oxidation of 
mineral compounds like those of Fe 2+ 

- decarbonation and deferrification in an acid medium can destroy 
minerals like amorphous silicates with precipitation of silica and 
elimination of Fe et al. (Frohlich, 1980). 

5.2.3 Brief guide to interpretation of the spectra 



General Principles 

In IR, transitions between the different energy levels are subject to rules 
of selection as absorption is linked to variation in the dipole moment of 



IR Spectrometry 147 

the molecules. In the case of polyatomic molecules, all predictable 
frequencies cannot be observed because energy levels are degenerated for 
example by symmetry in the molecule. It is thus, very difficult to 
accurately predict the frequencies of fundamental vibrations in complex 
structures like those of clays, though recent computer programs have 
enabled progress to be made. 

Tetrahedrons of silica and octahedral aluminium or magnesium form 
the basic units of clay minerals: a tetrahedral structure can produce four 
modes of vibrations, and an octahedral structure six, but these are not all 
active and can be modified by isomorphic substitutions or by the nature 
of the structural cations. 

The interpretation of a clay spectrum can be carried out after 
comparison with the spectrum of a "pure" substance of comparable nature 
in order to eliminate uncertainties caused by chemical variations in the 
composition and order-disorder state. The need for known standard 
spectra of reference implies each laboratory should record all results of 
studies on soil minerals to be used as references in addition to consulting 
available data bases. 

Qualitatively, it is first necessary to locate the intensity of the 
diagnostic absorption bands of minerals and to assign them to molecular 
groups and possibly to types of precise vibrations. The degree of 
sensitivity is satisfactory in the case of certain minerals that have intense 
bands (e.g. kaolinite, quartz, gibbsite, calcite). Quantities of the order of 
1% can be detected. 

For example, pure hydroxides and oxyhydroxides have a protonic 
environment that results in net vibrations of specific stretching; on the 
other hand, in the case of 2:1 and 2:1:1 minerals where chemical 
variations and isomorphic substitutions are frequent, displacement of the 
bands can occur; in this case it is useful to simultaneously use XRD 
analysis (cf. Chap. 4) and chemical analysis by selective dissolution 
(cf. Chap. 6) for secondary compounds of the soil, making it possible to draw 
up (silica of tetrahedrons) mtios that satisfactorily reflect the environment 

(alumina of octahedrons) 
of hydroxy Is. 

1:1 kaolinite has a Si0 2 -to-Al 2 3 ratio of 2 and presents surface hydroxyls 
that produce four frequencies of characteristic IR absorptions (Table 5.1). 

Smectites and micas have a ratio of approximately 3. As hydroxyls in 
internal positions are associated with different octahedral cations, the 
absorption bands are not uniform in the 3,600 cm" 1 area and displacement 
of the frequencies of hydroxyl stretching may be observed. The level of 
occupation of the octahedral sites (di- and tri-octahedral) can be 
determined by XRD analysis at line 060, but IR analysis can provide 
additional information. 



148 Mineralogical Analysis 

In the case of tri-octahedral minerals, the three sites are occupied and 
the axis of the OH bond of internal hydroxyl is perpendicular to the 001 
reticular plane of clays, whereas in di-octahedral minerals only two sites 
are occupied; the proton of internal hydroxyl is pushed back towards the 
empty octahedral sites and the spectrum is consequently deformed. 

Procedure 

The unknown spectra are manually broken up into fields and the bands of 
maximum intensity are selected along with the wavenumbers of the 
maxima to compare with reference data. This search can be automated 
with computerized data bases but in practice also requires the use of 
laboratory reference data and continuous consultation of the literature. 

In solid minerals, interpretation is mainly based on the frequencies of the 
external molecular group, as the detection of vibrations of the internal 
crystal is only significant in far IR. IR spectrometry accounts 
satisfactorily for the molecular groups in which the atoms are in a 
specific environment. Molecular structures with characteristic bands can 
be isolated. 

IR Absorption Bands in Phyllosilicates 

The apparent simplicity of soil minerals masks the complexity of 
absorption bands of the fundamental modes of vibration (Fig. 5.6). The 
bands are displaced as a function of the crystalline environment, of 
substitutions, etc. The frequency and assignment of the bands require 
very precise spectra to separate slight variations from phases that are 
often about 2 cm" 1 . For example, distinction of amorphous silica, opal, 
biogenic silica by means of the Si-O, Si-O-Si, Si-OH vibrations are of 
this order of magnitude. 



In the 3,700- 
3,400 cm" 1 and 
950-600 cm" 1 
zones 



protons in hydroxyls groups, even if there is no 
long-distance molecular structure, which is 
useful in the case of "amorphous" substances 

the active modes of tetrahedrons and 
octahedrons: OH oscillation, dichroism of OH 
sites in di-octahedral minerals 



IR Spectrometry 



149 



displacement of the absorption bands as a 
function of the nature of the octahedral cations, 
effect of exchange (e.g. interaction of structural 
OH with interlayer cations) 

separation of the stretching vibrations of non- 
bound water allows better distinction of the 
halloysites disturbed by interfoliaceous water 
molecules 



In the 1,100-500 
cm" 1 zone 



vibrations of the silicate anion that are 
considered as the spectral print of clays, slightly 
coupled with vibrations of other structures 
(silica-oxygen bonds), Si-0 stretching towards 
1,000 cm" 1 , Si-0 deformation towards 500 cm" 1 ; 

isomorphic substitutions of tetra and octahedral 
minerals can cause bands in far IR 



In the <400 cm" 1 
zone 



Al 3+ of octahedrons and oxygen bonds of 
adjacent layers 

vibrations of exchangeable cations balancing the 
charges in interfoliaceous spaces of clays and 
substitution of exchangeable cations 



Taking the example of the adsorption bands of a common clay, 1 : 1 
kaolinite (Table 5.1), it is possible to observe: 

- a similar configuration of the bands of proton vibration (stretching of 
external and internal hydroxyls) with respect to crystallinity; 

- a level of disorder that is detectable by the coalescence of the 3,669- 
3,653 cm" 1 doublet; 

-hydration water that is easily differentiated by deuteration and the 
appearance of a HOH band towards 1,630 cm" 1 



150 



Mineralogical Analysis 



-1,110, 1,036 and 1,010 bands corresponding to stretching vibrations 

characteristic of SiO. 

In the case of Halloysites, widening of the bands can be observed 
between 3,700 and 3,600 (OH stretching) because of the structural 
distortion caused by variable hydration. Bands 795 and 758 cm" 1 are of 
about the same intensity in kaolinite whereas in halloysites band 795 cm" 1 
is very weak. 



Table 5.1. IR absorption bands of well-crystallized kaolinite 
AI 2 03,2Si02,2H 2 0, <2|jm KBr pellet 



band 


Vibrations 




band 


Vibrations 




(s cnrf ) 






(s cnrf ) 






3,695 




external OH 


690 


- 


Mg/AI 


3,669 




external OH 


538 


deformation 


SiO 


3,653 


stretching 


external OH 


470 


+ 


+ 


3,619 




internal OH 


430 






3,410- 




OH hydration 


364 


vibrations 


Al/Mg 


3,450 a 










octahedrons 


1 ,630 a 


deformation 


HOH 
hydration 


345 


miscellaneous 




1,106- 


stretching 


out layer SiO 


275 






1,112 












1,036 




SiO... 


195 






1,010 




SiO... 








938 


Deformation 


external OH 








912 




internal OH 









a water of hydration (towards 3,450 cm 1 ) can be distinguished from the OH of 
structural hydroxyls by the presence of a HOH band towards 1 ,630 cm" 1 



IR Spectrometry 



151 



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152 Mineralogical Analysis 

5.2.4 Quantitative Analysis 



Definition 

In mineralogy, improvements in quantitative analysis using IR have 
accompanied improvements in FTIR equipment, and quantitative analysis 
is an important tool for the study like pedogenic chronosequences, flows 
and sedimentary series (e.g. paleohydrology, paleoclimatology) or 
watershed functioning. It is always interesting to observe a mineral in its 
original state without pretreatment, with the exception of grinding in a 
non-aggressive liquid medium to reduce granulometry to 1-2 |Lim, then 
drying. 

The relations between the intensity of absorption of IR radiations and 
the concentration of mineral species are controlled by the law of Beer- 
Lambert which is applicable to solid media (Keller and Picket, 1949, or; 
Duyckaerts, 1959). However, this law is not always applicable to soil 
minerals for which the characteristic bands are not quite separate and 
often insufficiently homogeneous. The order-disorder states affect the 
band widths, and grain sizes influence band intensity. In this case, the 
limitations are obvious and the photometric response is seldom linear: 
the absorbance of a band is then no longer proportional to the concentra- 
tion, and quantification cannot be correctly carried out as it can for gas or 
organic liquids. 

Since standard minerals (with structure, crystallinity, chemical 
composition and particle size similar to that of the samples) do not exist, 
only relative quantification can be obtained using this method, 
nevertheless it is possible to measure variations in the composition of 
minerals in a profile or toposequences with acceptable precision. 

For certain minerals with a relatively characteristic spectrum and a 
well defined base line such as some quartz, kaolinite, carbonates, the 
levels of detection will be about 1-2%. Purification by sedimentation 
allows concentration and simplification of the spectra. The state of 
crystalline order can cause significant variations, for example in 
kaolinites when structural OH between 3,700 and 3,600 cm" 1 are used. 

The preparation of the sample is particularly important for quantitative 
analysis. Maximum absorption (without saturation) and minimum 
dispersion are required. The isogranulometric size of the particles must be 
less than 2 |um. The homogeneity of the discs must be perfect, the 
components should be dried at each stage of preparation or storage, and 
the thickness of the sample must be constant at less than 1 mm. 



I R Spectrometry 153 

Rigorous standardization of the procedures makes it possible to 
optimize measurements of soil mineral components without depending 
completely on the degree of crystallinity of powders as in XRD (cf. Chap. 
4). Some minerals with short-range organization that are "amorphous" to 
X-rays, for example some aluminosilicates, crypto crystalline compounds 
(e.g. allophane, imogolite), and some forms of silica and of iron (e.g. 
ferrihydrites) can be quantified in this way. 

Procedure 

Overview 

The procedures for qualitative analysis (cf. Sect. 5.2.1 and 5.2.2) are 
applicable here, and particular care should be taken to: 

-desiccate the samples and binders at 105°C for one hour before 
weighing precisely (10~ 6 g); a temperature of 40°C can be used for 
heat-sensitive substances 

- grind the clay samples to less than 1-2 |um with an agate mortar or 
preferably with a tightly adjusted cooled vibratory grinder with agate 
balls in wet medium (in the presence of ethanol or of acetone); after 
drying, all the particles should pass through a 0.1 mm sieve; fractions 
that are quantitatively isolated by ultracentrifugation (cf. Chap. 3) can 
also be used 

-weigh a quantity of clay allowing transmittance ranging between 
approximately 20% and 70% for the whole band range of the spectrum 
(qualitative tests make it possible to fix the exact proportions between 2 
and 5 mg of sample for 1 g of binding agent); mix with the appropriate 
binding agent (often KBr) and homogenize with a vibratory mixer with 
agate balls for 5 min 

- using 1 g of the above mixture, make three discs with a diameter of 13 
mm each by weighing the same quantity of mixture (300-330 mg) to 
obtain a constant optical pathway of one mm or less; transfer to the 
pelletizer under vacuum (Fig. 5.5) and apply a pressure of 10 tons cm" 2 
for 2-3 min; the discs should be transparent, smooth, show no surface 
defects, be of constant thickness and present regular dispersion of the 
clay particles in the binding agent (this should be checked under the 
microscope); they should be handled using a forceps and stored in a 
desiccator with phosphoric anhydride; prepare the calibration discs in 
the same way 

- measure the absorbance of the discs made of binding agent (for the 
blank assay), sample disks and pure or complex calibration discs made 



154 



Mineralogical Analysis 



according to the laboratory reference, which enable calibrations with 
variable proportions of minerals. 

Sediments 

Frohlich (1980, 1989) recommended grinding with an agate ball to less 

than 2 |Lim in a cooled inert liquid medium. The fineness of grinding 

should be checked under the microscope. Optimal dilution is around 

0.25%. 

-Prepare 1 g of mixture: 2.5 mg of sediment (precision 10" 5 to 10" 5 g) 

and 997.5 mg of KBr 
- homogenize carefully with a vibrating grinder with an agate ball; 
-press 300 mg of the mixture for 2 min in a 13 -mm diameter mould 

under vacuum. 






Fig. 5.7. Determination of T :good base line (a, b), approximation (c). 



IR Spectrometry 155 

The thickness of the KBr 1 disc should be 0.83 mm and represent the 
constant optical pathway in all measurements. The disc should be smooth 
and transparent and should contain 0.75 mg of sample. 

Calculations 

Any absorbance due to the thinner (KBr) must be subtracted from total 
absorbance (KBr+sample). The transmission (and by conversion 
absorbance A) of the substance is measured on the spectrum starting from 
the base line. This line is often difficult to define for complex mixtures 
(Fig. 5.7) and requires approximations (effect of matrix, interference 
between bands, etc.). The relative error is often about 5%, and can be 
improved for certain minerals. 2 Calibration using "pure" minerals or 
mineral mixtures with a composition close to that of the samples makes it 
possible to plot curves of absorbance =/(mass of mineral). 

One gram of mixture makes it possible to estimate repeatability on 
three discs weighing 300 mg. 

Remarks 

Strong orientation of minerals in the disc can generate errors. In 2:1 
clays, variations in intensity of the stretching bands of hydroxyls can 
result from tri-octahedral components. With micas oriented perpendicular 
to the beam, only modes of vibration parallel to plane b will appear 
(Phlogopite). Conversely, in kaolinite, the intensity of the 3,619 cm" 1 
band is independent of the orientation (internal hydroxyl directed towards 
the vacant octahedral position). 

If titrations are carried out with a traditional dispersive apparatus, the 
resolution can be improved by finer slits but the energy will be weaker. 
As the width of the slits is not constant throughout the spectrum, care 
should be taken that the slits are not too wide, because the signals could 
be deformed and the law of Beer-Lambert would then not apply. 



Density of KBr: 2.75 

In spite of the use of reference minerals, the great variability of the absorption 
bands as a function of the chemical structure often results in insurmountable 
difficulties in calibration of 2:1 and 2:1:1 minerals. In this case, it is possible to 
obtain only semi-quantitative measurements that allow identification of changes in 
a profile, the mineral tracer being used as standard of comparison at a given 
level. 



156 Mineralogical Analysis 

5.3. Other IR techniques 



5.3.1 Near-infrared spectrometry (NIRS) 



Principle 

Vibrations of light atoms that have strong molecular bonds with protons 
(N, C or O) are used to analyse organo-mineral compounds or organic 
matter. When the bonds are weak and the atoms are heavy, it can be 
difficult to detect and quantify the vibration phenomena. 

Wide bands are reproducible but are influenced by penetration of the 
radiation, and thus sensitive to the size of the particles and to moisture. 
Fine grinding is usually required to obtain particles of the same size and 
to reduce the background noise as much as possible. But acceptable 
results can be obtained with materials that are not finely ground (D. 
Brunet, IRD Montpellier, France, personal communication). Bond 
vibrations cause a response that depends on the number of molecules 
present and on their environment, this response then enables quantification 

The bands in the near-infrared field are more widely spaced than in 
medium and far IR, which limits the phenomena of overlapping. The first 
derivative of the signal can be used to improve precision. 

Measurements are made either by transmission-absorption, or by 
diffuse emission-reflectance (NIRA-DRIFT) 3 on powders using wavelengths 
ranging from 1,000 to 2,500 nm (wavenumbers from 10,000 to 4,000 cm" 1 ) 
in certain cases such as soil litter (water, protein content, total nitrogen, 
sugars, etc.), or on liquids using immersed optical fibres. 

Material 

Measuring equipment with diffuse reflectance IR is somewhat different 
from IR spectrometry using the transmission-absorption mode (cf. 
diagram in Fig. 5.8). The optical elements are made of quartz. They allow 
the complete near-IR spectrum to be acquired in a few seconds. 



3 NIRS = near-infrared reflectance spectrometry 
NIRA = near-infrared analysis 
DRIFT = diffuse reflectance IR with fourier transform 



IR Spectrometry 



157 



Method 

This non-destructive method requires fine grinding, but does not require a 
reagent, or weighing or measurement of volume. Measurements are rapid 
(=30 s) and the unit cost of the measurement is low. 

Measurements depend on the physical factors that affect reflectance, 
i.e. particle size (reflectance, refraction, diffraction), their distribution 
(heterogeneity) and the distribution of the vacuums (compaction and 
induced orientations). 



Diaphragm 



Source 
(tungsten lamp) 



Filter 

Monochromator 

Interferometer 



Data 

acquisition 
and treatment 



PbS 
Detector 




Thermostated 
rotating cup 
(sample) 

Fig. 5.8. Diagram of a near-infrared spectrometer (NIRS) 1: oscillating mirror 
allowing adjustment of the incidental beam on the sample and improving 
reflectance on the walls of the integration sphere. All possible angles 
must be represented starting from the normal. The total flow from the 
source that excites the sample is concentrated by quartz lenses. 2: 
Integration sphere allowing the effect of variations in particle size to be 
decreased with collection of the most intense radiation but rejection of 
specular components 



Current NIRS systems have been greatly improved by progress in 
chemometric software which enables calculations that were previously 
impossible. Quantitative analysis is based on multivariate calibration 
using all the spectral information, not only absorbance at a given 
wavelengths but absorbance at all wavelengths of the spectrum. These 
calibrations use a wide range of methods of calculation, especially 



158 Mineralogical Analysis 

principal component analysis (PCA), regression on principal components 
(RPC), partial least square (PLS) regression and multiple linear 
regression (MLR). The software includes help in choosing the best 
method of calibration, even if the choice is still not always easy 
(Dardenne et al., 2000). 

This method is suitable for organic analysis but was extended to many 
different measurements on soils. Chang et al. (2001) used RPC 
calibration for FTIR determination of moisture content, total C, total N, 
CEC, sand content, silt content, clay content, macro-aggregation, 
potentially mineralisable N, C biomass, total respiration rate and basal 
respiration rate of soils. The only condition is the need to compile a 
database of soil references for calibration. For a given variable, 
calibration consists of (1) obtaining a measurement value by a reference 
method for all soil references (preferably including a wide range of 
concentrations of the given variable), (2) obtaining NIR spectra on the 
same soil references, (3) calculating and plotting the straight line of 
multivariate calibration with the value measured using the standard 
method in the x-coordinate and predicted value by NIRS in the 
^-coordinate. After calibration, the measurement of an unknown sample 
is very rapid: its real concentration in the x-coordinate can be deduced 
from its spectral data in the ^-coordinate. But the apparently universal 
application of the method is not quite true. Calibration is always possible 
but not always significant (e.g. variability of the calibration curves 
obtained by Chang et al., 2001). Which soils to choose for the soil 
references (all soil types, or a given soil type)? What type of soil 
preparation? The complementary bibliography at the end of this chapter 
lists a few additional applications of NIRS in soil and litter studies. 

5.3.2 Coupling Thermal Measurements and FTIR Spectrometry 
of Volatile Products 

Measurements are taken during TGA-DTA 4 and enable determination of 
the nature of the gas products that appear during heat decomposition of 
the sample (EGD or EGA) 5 The analyses are carried out by Fourier 
transform infra-red spectrometry (FTIR) by transmission or absorption in 
time of flight, as a function of the temperature and heating time. 

This dynamic technique enables real-time monitoring of the chemical 
or physicochemical conversions that take place during the rapid heating 



4 DTA = Differential thermal Analyse, TGA = Thermo Gravimetric analyzes, cf. Chap. 7. 

5 EGD = Evolved Gas Detection, EGA = Evolved Gas Analysis. 



I R Spectrometry 159 

of the sample (controlled thermolyses or pyrolyses of organic or 
inorganic material is possible). A rise in temperature at moderate speed 
makes it possible to detect unstable radicals and molecular 
fragmentations by linking them to variations in mass and temperature 
(fusion, exo- and endothermic reactions, decomposition of mineral- 
carbonates, N, C, S, H compounds, oxidation, reduction, transfers of 
protons etc.). 

Pressure is a variable that affects sublimation and evaporation. 
Pressure can be modified by too rapid decomposition of an unstable 
product, but, depending on the temperature, can also cause molecular 
synthesis. Under low pressure, the most reactive gases diffuse quickly, 
avoiding possible recombination. Working under argon atmosphere at 
low pressure is generally recommended. But working under controlled 
atmosphere can highlight redox phenomena or, on the contrary, avoid 
them. 

Additional information can be collected by selecting a heating rate 
between 20 and 400°C min" 1 depending on the speed of evacuation of the 
gas produced and on its detection or rapid titration before further gaseous 
reactions occur. 

5.3.3 Infrared Microscopy 

FTIR analysis is possible on microsamples measuring from 20 to 500 urn. 
The IR microscope (cf. Chap. 8) consists of lenses with a Cassegrain 
mirror coupled with a high sensitivity MCT 6 detector cooled with liquid 
nitrogen. It is possible to work with either transmission or reflectance. 
The resolution is approximately 8 cm" 1 depending on the quality of the 
materials and the number of accumulations of spectra. 

It is also possible to use Raman spectrometry where the source of 
excitation is a monochromatic laser emitting in the red band to avoid the 
effects of fluorescence which occurs in the presence of certain organic 
materials. Quantities of the order of a pico and even of a femtogramme 
can be detected in this way. 

5.3.4 Raman scattering spectroscopy 

Interest 

Raman spectroscopy has not been widely used in earth science because 
dispersive equipment is very expensive and the performance is often 



6 MCT = mercury, cadmium, tellurium. 



160 Mineralogical Analysis 

insufficient due to the difficulty of obtaining a selection of wavelengths 
with high resolution. 

Progress in electronics has made it possible to design very sensitive 
detectors, very selective monochromators, and powerful monochromatic 
lasers, and to use FTIR spectrometers thus making this technique 
accessible and complementary to other IR spectrometries. Data 
processing enables very rapid treatment of the spectra. 

This technique makes it possible to supplement the information 
obtained in transmission-absorption IR spectrometry, as certain 
vibrations are only active in one of the two techniques, or their intensity 
differs because of the rules of selection. The symmetrical vibration bands 
are stronger in Raman spectroscopy and the asymmetrical vibrations are 
stronger in IR spectroscopy. 

The study of certain molecular structures that are difficult to 
differentiate such as rutiles, anatases and brookites is possible on 
microsamples. The nature of the chemical bonds and the orientation of 
OH groups can be determined without obstruction by any interstitial 
water that may be present. The method is not destructive and does not 
require complex preparation. It is possible to work on powder, even wet 
powder, which is impossible with other IR techniques. 

Principle 

Raman spectroscopy is based on the inelastic scattering of IR 7 radiation 
with IR secondary emission at beat frequencies. The spectra are 
composed of fine lines which require a high resolution apparatus: 

- phenomena of fluorescence induced by the electronic transitions can 
disturb the spectra and mask the Raman signal if an excitation laser that 
emits in the visible spectrum (488 nm) is used; excitation by Nd:YAG 
laser emitting at 1,064 nm, the frequency corresponding to a zone of 
little occupied electronic transition, generally does not generate 
fluorescence, and coupling with a good-quality FTIR spectrometer 
eliminates difficulties due to insufficient resolution; 

- when a monochromatic radiation beam strikes a sample, a weak fraction 
of the re-emitted radiation displays modified frequencies that reflect the 
vibration frequencies of the sample. This fraction is measured in Raman 
spectrometry; the unchanged radiation fraction (Rayleigh elastic 
scattering) has to be removed by filtering. 



7 Raman spectroscopy in visible or UV radiation can cause photodecomposition of 
the sample as well as thermal damage. 



IR Spectrometry 161 

Apparatus 

The basic apparatus is a FTIR spectrometer equipped with an 
interferometer coupled with data acquisition and processing software. It 
should have an external window to attach a Nd-YAG laser irradiation, a 
chamber for sample powders allowing irradiation modes of 90° and 180°, 
a spectral filtration module and if necessary a specific detector. A Raman- 
FT microscope and accessories for Raman studies under very high 
pressure (diamond anvils) can be added to the FTIR spectrometer. 

The laser for excitation of the atoms and molecules must be 
monochromatic or adjustable in wavelength (for better selectivity). It 
must provide strong intensity (sensitivity of measurements), coherent 
radiation (spatial and temporal quality), and finally, if the beam is 
transported by optical fibre, low divergence. 

IR and Raman spectroscopy can be supplemented with other 
techniques for the study of structure (e.g. NMR, EXAFS). In spite of the 
development of these techniques, the IR and Raman methods remain 
competitive (ease of handling, reasonable cost of equipment) and allow a 
sufficiently detailed approach of the structure (including vacancies and 
substitutions, nature of the bonds in molecules) and of its consequences 
for rheology, for example, or for the study of pedogenesis phenomena 
that occur during weathering (e.g. coverings, interactions with the 
surface, adsorption of molecules). However, the quantification of the 
phases is often delicate if not impossible, because of the problems of 
orientation of clays and incomplete spectrum. 



References 

Chang C.-W., Laird DA, Mausbach M. et Hurburgh CRJr (2001) Near-Infrared 

Reflectance Spectroscopy-Principal Components regression analysis of 

soil properties. Soil Sci. Soc. Am. J., 65, 480-490 
Dardenne P, Sinnaev G et Baeten V (2000) Multivariate calibration and 

chemometrics for near infrared spectroscopy: which method. Journal of 

Near Infrared Spectroscopy, 8, 229-237 
Duyckaerts G (1959) The infra red analysis of solid substances. Analyst, 84, 201-214 
Farmer VC et Palmieri F (1975) The characterization of soil minerals by Infrared 

spectroscopy. In: Soil components - 2 - Inorganic components, 

Gieseking JE ed., Springer, 573-670 
Frohlich F (1980) Neoformation de silicates ferriferes amorphes dans la 

sedimentation pelagique recente. Bull Mineral, 103, 596-599 
Frohlich F (1989) Les silicates dans l'environnement pelagique de l'ocean 

indien du cenozoi'que. Memoire Museum National d'Histoire Naturelle, 

Paris, XL VI, 206p 



162 Mineralogical Analysis 

Keller WD et Pickett FE (1949) Absorption of IR radiation by powdered silice 

minerals. Am. Miner., 34, 855-868 
Liu LG et Mernagh TP (1992) Phase transitions and Raman spectra of anatase 

and rutile at high pressures and room temperature. Eur. J. Mineral, 4, 

45-22 
Nail SL, White JL et Hem SL (1976) IR studies of development of order in 

aluminium hydroxide gels. J. Pharm. Sci., 65, 231-234 
Nyquist RA et Kagel O (1971) Infrared spectra of inorganic compounds., 

Academic Press, New York 
Stubican V et Roy R (1961) Infrared spectra of layer silicates. J. Am. Ceram. 

Soc, 44, 625 
Wada K (1966) Deuterium exchange of hydroxyl groups in allophane. Soil Sci. 

Plant Nutr., 12, 176-182 
Weir CE Lippincott ER Van Valkenburg A et Bunting EN (1959) Infra-red 

studies in the 1 and 15 microns region to 30 000 atmospheres. J. Res. 

Natl. Bur. Stud., 63 A, 55 



Chronobibliography 



Tuddenham WM et Lyon R.P (1960) Infrared techniques in the identification 

and measurement of minerals. Anal. Chem., 32, 1630-1634 
Mitchell BD Farmer VC et Mc Hardy WJ (1964) Amorphous inorganic 

materials in soils. Academic Press. Adv. Agron., 16, 327-383 
Hayashi H et Oinuma K (1965) Relationship between infrared absorption spectra 

in the region of 450-900 cm -1 and chemical composition of chlorite. 

Am. Miner., 50, 476-483 
Hayashi H et Oinuma K (1967) Si-0 absorption band near 1000 cm" 1 OH 

absorption bands of chlorite. Am. Miner., 52, 1206-1210 
Russell JD, McHardy WJ et Fraser A.R (1969) Imogolite: a unique alumino- 

silicate. Clay Miner., 8, 87-99 
Wada K et Greenland DJ (1970) Selective dissolution and differential infrared 

spectroscopy for characterization of amorphous constituents in soil 

clays. Clay Miner., 8, 241-254 
Conley RT (1972) Infra-red spectroscopy. Allyn-Bacon, 2nd. Edition 
Fieldes M, Furkert R.J et Wells N (1972) Rapid determination of constituants of 

whole soils using IR absorption. N. Z. J. Sci., 15, 615-627 
Miller RGT et Stace BC (1972) Laboratory methods in Infrared spectroscopy., 

Heyden and Son 
Farmer VC (1974) The Infrared spectra of minerals. Minerals Sci. (London). 
Stepanov IS (1974) Interpretation of the IR spectra of soils. Pochvovedenie, 6, 

76-88 
Gadsden JA (1975) Infrared spectra of minerals and related inorganic 

compounds., Butterworth 



IR Spectrometry 163 

Griffiths PR (1975) Chemical infrared four ier transform spectroscopy., Wiley, 

New York Chemical Analysis, 43 
Brame EG, Grasselli JG (1976) Infrared and Raman spectroscopy., Marcel 

Dekker, 1A 
White JL, Nail SL et Hem SL (1976) Infrared technique for distinguishing 

between amorphous and crystalline aluminium hydroxide phase. 

Proceedings. 7th Conference, clay Mineral Petrology (Czechoslovakia), 

51-59 
Marel HW, Van der et Beutelspacher H (1976) Atlas of infrared spectroscopy of 

clay minerals and their mixtures., Elsevier Amsterdam 
Proshina NV (1976) Use of infrared spectroscopy for identification of soil 

samples. Nauch. dokl. Vsshei Shk., Biol. Naudi, 3, 1 14-1 18 
Brame EG, Grasselli JG (1977) Infrared and Raman spectroscopy., Marcel 

Dekker, IB, 1C 
Hlavay J, Jonas K, Elek S et Inczedy J (1977) Characterization of the particle 

size and the cristallinity of certain minerals by infrared spectrophotometry 

and instrumental methods. I - Investigations on clay minerals. Clays 

Clay Miner., 25, 451-456 
Hlavay J, Jonas K, Elek S et Inczedy J (1978) Characterization of the particle 

size and the crystallinity of certain minerals by infrared spectrophotometry 

and other instrumental methods. II-Investigation on quartz and feldspar. 

Clays Clay Miner., 26, 139-143 
Ferraro JR et Basile LJ (1978) Fourier transform infrared spectroscopy. 

Applications to chemical systems., Academic, New York, vol. 1 
Slonimskaya MV, Besson G, Dainyak LG, Tchoubar C et Drits VA (1978) 

Interpretation of the IR spectra of celadonites and glaucomites in the 

region of OH-streching frequencies. Clay Miner., 21, 377-388 
Smith AL (1979) Applied infrared spectroscopy: fundamentals, techniques and 

analytical problem-solving., Wiley, New York, vol. 54 (chemical 

analysis). 
Farmer VC (1979) The role of infrared spectroscopy in a soil research institute: 

characterization of inorganic materials. Eur. Spectrosc. News, 25, 25-27 
Ferraro JR et Basile LJ (1979) Fourier transform infrared spectroscopy. 

Applications to chemical systems., Academic, vol. 2 
Hlavay J et Inczedy J (1979) Sources of error of quantitative determination of 

the solid crystalline minerals by inrared spectroscopy. Acta Chim., 

(Budapest), 102, 11-18 
Olphen H Van et Fripiat JJ (1979) Data handbook for clay materials and other 

non-metallic minerals., Pergamon 
Martin AE (1980) Infrared interferometric spectrometers. In Vibrational spectra 

and structure, Durig J.R. ed., Elsevier, Amsterdam, vol. 8 
Pouchert CJ (1981) The Aldrich library of infrared spectra., Aldrich Chemical 

Co, 1850 p 
Shika A, Osipova NN et Sokolova TA (1982) Feasibility of characterizing the 

mineralogical composition of soils by infrared spectrophotometry. 

Moscow Univer. Soil Sci. Bull, 37, 34-40 



164 Mineralogical Analysis 

Theng BKG, Russel M, Churchman GJ et Parfitt RL (1982) Surface properties 

of allophane, halloysite and imogolite. Clays Clay miner., 30, 143-149 
Ferraro JR et Basile LJ (1983) Fourier transform infrared spectroscopy. 

Applications to chemical systems., Academic, New York , vol. 3 
Fysh SA et Fredericks PM (1983) Fourier transform infrared studies of 

aluminous goethites and hematites. Clays clay Miner., 31, 377-382 
Velde B (1983) Infra-red OH-stretch bands in potassic micas, talcs and 

saponites: influence of electronic configuration and site of charge 

compensation. Am. miner., 68, 1 169-1 173 
Gillette PC et Koenig JL (1984) Objective criteria for absorbance subtraction. 

Appl. Spectrosc, 38, 334-337 
Kosmas CS, Curi N, Bryant RB et Franzmeier DP (1984) Charactrization of iron 

oxide minerals by second-derivative visible spectroscopy. Soil Sci. Soc. 

Am. J., 48, 401-405 
Prost R (1984) Etude par spectroscopic infra-rouge a basse temperature de 

groupes OH de structure de la kaolinite, de la dickite et de la nacrite. 

Agronomie, 4, 403^06 
Kodama H (1985) Infrared spectra of minerals. Reference guide to identification 

and characterization of minerals for the study of soils. Res. Branch, 

Agric. Can. Tech. Bull., IE 
Mulla DJ, Low PF et Roth CB (1985) Measurement of the specific surface area 

of clays by internal reflectance spectroscopy. Clays Clay Miner., 33, 

391-396 
Keller RJ (1986) The Sigma library ofFT-IR spectra., Sigma chemical Co, vols. 

1-2, 2894 p 
Griffiths et Haseth PR (1986) Fourier transform infrared spectrometry., 

Chemical Analysis Series, Vol. 83, Wiley New York, 672 p 
Russel JD (1987) Infrared spectroscopy of inorganic compounds. In Laboratory 

methods in infra-red spectroscopy, Willis H.ed., Wiley, New York 
Johannsen PG, Krobok MP et Holzapfel WB (1988) High-pressure FT-IR 

spectrometry., Bruker report, 39-43 
Pouchert CJ (1989) The Aldrich library of FT-IR Spectra., Aldrich Chemical Co, 

vols. 1-3, 4800 p 
Mottana A et Burragato F (1990) Absorption spectroscopy in mineralogy., 

Elsevier, Amsterdam, Oxford, New York, Tokyo, 294 p 
Delvigne JE (1998) Atlas of Micromorphology of mineral alteration and 

weathering. The Canadian Mineralogist, special publication 3, Ottawa 

et IRD (ex-Orstom), Paris 
Silverstein RM et Webster FX (1998) Spectrometric Identification of organic 

compounds. Wiley New York, 482 p 
McHale JL (1999) Molecular spectroscopy., Prentice-Hall, London, Sydney, 

Toronto, 463 p 
Gillon D, Joffre R et Ibrahima A. (1999) Can litter decomposability be 

predicted by near infrared reflectance spectroscopy. Ecology, 80, 175— 

186 
Confalonieri M, Fornasier F, Ursino A, Boccardi F, Pintus B et Odoardi M 

(2001) The potential of near infrared reflectance spectroscopy as a tool 



IR Spectrometry 165 

for the chemical characterisation of agricultural soils. J. Near Infrared 

Spectrosc, 9, 123-131 
Joffre R, Agren GI, Gillon D et Bosatta E (2001) Organic matter quality in 

ecological studies: theory meets experiment. Oikos, 93, 451-458 
Fearn T (2001) Standardisation and calibration transfer for near infrared 

instruments: a review. J. Near Infrared Spectrosc., 9, 229-244 
Ludwig B et Khanna PK (2001) Use of near infrared spectroscopy to determine 

inorganic and organic carbon fractions in soil and litter. In Assessment 

methods for soil carbon, Lai R, Kimble JM, Follet RF et Stewart BA 

ed., Lewis, UK 
Ozaki Y, Sasic S et Jiang JH (2001) How can we unravel complicated near 

infrared spectra? - Recent progress in spectral analysis methods for 

resolution enhancement and band assignments in the near infrared 

region. J. Near Infrared Spectrosc, 9, 63-95 
Reeves J B et McCarty G W (2001) Quantitative analysis of agricultural soils 

using near infrared reflectance spectroscopy and a fibre-optic probe. J. 

Near Infrared Spectrosc, 9, 1, 25-34 
Tso, Ritchie GE, Gehrlein L et Ciurczak EW (2001) A general test method for 

the development, validation and routine use of disposable near infrared 

spectroscopic libraries. J. Near Infrared Spectrosc, 9, 165-184 
Fidencio PH, Poppi RJ et de Andrade JC (2002) Determination of organic matter 

in soils using radial basis function networks and near infrared 

spectroscopy. Anal. Chem. Acta., 453, 125-134 
Couteaux MM, Berg B and Rovira P (2003) Near infrared reflectance 

spectroscopy for determination of organic matter fractions including 

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1587-1600 
Brown DJ, Shepherd KD, Walsh MG, Dewayne Mays M and Reinsch TG 

(2005). Global soil characterization with VNIR diffuse reflectance 

spectroscopy. Geoderma, doi:10.1016/j.geoderma.2005.04.025 



Mineralogical Separation by Selective 
Dissolution 



6.1 Introduction 



6.1.1 Crystallinity of Clay Minerals 

Mineralogical characterization of cryptocrystalline minerals or minerals 
with short-range atomic arrangement (Fe, Al, Si, Mn, Ti, P) is essential to 
understand the geochemical and pedochemical phenomena that occur 
during the weathering of primary minerals, as well as to explain the 
evolution and the relative stability of the systems and the kinetics of 
chemical soil processes. These substances can represent the transition 
stage between the crystalline parent rock and secondary minerals, and are 
often regarded as tracers of evolution. The soil is an open system, i.e. it is 
able to exchange energy and matter with the outside. Most reactions 
occur under non-equilibrium conditions, and transitory states depend on 
aqueous or gas flows (chemical reactions at the solid-liquid and liquid- 
liquid interface), on relaxation times (particle diffusion, transfer of 
matter, etc.), and of course, on microbial activity. 

The individual accumulation of these substances, or their deposit in a 
fine layer of coating, modifies the activity of the structural sites of 
crystalline materials, and can inhibit the movement of ions, neutralize 
charges, or cause substitutions in the lattices. Gels, oxides and 
oxyhydroxydes, and aluminosilicates can develop charges (some of 
which are amphoteric). The high level of reactivity induced by their state 
of division allows adsorption of cations and anions. They can be 
neutralized by organic substances. These reactions confer greater 
resistance to weathering and to microbial action. Thus, the ultimate 
purpose of analysis of non-crystalline products may be soil genesis, and 
also: 



168 Mineralogical Analysis 

- Soil taxonomy (through processes of podzolisation, andosolisation, 
laterisation, etc.) 

- Soil mineralogy, mineralogical balances, purification before using other 
techniques (in particular methods that require the elimination of 
paramagnetic elements) e.g. ESR, EXAFS, Mossbauer, XRD, FTIR, 
SEM, EDX, WDX, TEM-HR, STEM 1 

- The study of the physical and chemical properties of the soil, studies of 
soil fertility (Fe deficiencies, P fixing, Al 3+ toxicity, transport of heavy 
metals, destruction of the interparticle cements, aggregation factors, 
etc.) 

Identification of non-crystalline substances requires more than one 
method: XRD is not very useful with gels because if the quantity of gel is 
significant, or the early stages of development of a long-range crystalline 
structure are concerned, only broad bands will be obtained. Chemical 
dissolution methods are not sufficiently selective in mineralogy, as their 
action is based on acid, base, reducing, or complexing reagents, and 
consists for example in: 
-Breaking electrostatic (e.g. exchange reactions, Al 3+ bridges) or 

coordination bonds (e.g. Fe 3+ bridges) 

- Causing ionization of functional groups (organic matter) 

Dissolution must not only enable extraction of the different phases that 
are amorphous to X-ray but also: 

- Minimize chemical modifications (relative instability of the products to 
be extracted compared to the soil matrix, and avoid attacks of the clay 
lattices and primary minerals) 

- Limit hydrolysis of the extracted products 

- Avoid molecular rearrangements in the liquid phase (nucleation) 

- Maintain the extracted products in solution 

- Prevent the creation of chemical barriers (insoluble precipitate under 
the influence of the reagents) and the neo-formation of solid products 



1 



ESR electron spin resonance; EXAFS extended X-ray adsorption fine structure; 
XRD X-ray diffraction; FTIR Fourier transform infra-red; SEM scanning 
electron microscopy; EDX energy dispersive X-ray; WDX wavelength 
dispersive X-ray; STEM scanning transmission electron microscopy. 



Selective Dissolution 169 

All the extractions (e.g. single reagent or multiple reagents, single or 
sequential extractions) depend on thermodynamic constraints: 

- Ion activity, pH, concentration of the reagents, soil/reagent ratio, order 
of application of the reagents 

-Time factors, kinetics of extraction, stirring velocity, duration of 
contact, ageing of the gel 

- Temperature 

- Photo lytic energy (UV catalyses of the chemical reactions) 

Initially the rate of mineralogical extraction is often significant, but 
subsequently levels off; it is also linked to the size of crystallites and 
perfection of crystallinity (defects, degree of disorder), or to the nature 
and the concentration of the elements in the liquid phase. Agreement with 
other studies, reproducibility and reliability will thus depend on the 
extraction procedures used. Unless justified by the need to adapt to 
specific problems, any modification in the procedure (proportion or 
concentration of reagents, time of contact, etc.) can cause serious errors 
in evaluation. The required degree of selectivity of mineral extraction can 
be obtained only by comparing different extractions that have been 
carefully purified by ultracentrifugation, and by chemical measurement 
(1) on the liquid phase containing the extracted products (congruent and 
incongruent reactions) and (2) on the solid phase (e.g. differential XRD, 
SEM, and EDX). Automatic calculation and interpretation can be 
performed with a limited number of reliable methods and this makes it 
possible to quantify each phase and to establish precise and reproducible 
geochemical balances. 

6.1.2 Instrumental and Chemical Methods 

Measurement by X-ray diffraction works well for atomic lattices with 
long-range organization, but for substances with short-range arrangement 
without ordered superstructure, XRD gives flat, unusable spectra 
(Fig. 6.1). This is why substances presenting this type of flat X-ray 
spectrum are referred to as amorphous substances. 

Progress in instrumental methods has now made it possible to specify 
the nature of the phases and to determine their arrangement more 
precisely. The crystalline state is characterized by the periodic repetition 
of an atomic structure along three non-coplanar directions of space 
(Maziere 1978). The use of "non-crystalline" or crypto-crystalline 
substances, rather than paracristalline, is now allowed (amorphous, 
without structure; crypto, masked structure; para, almost a structure). It 
applies to the solids whose structure does not present a repetitive nature 
at long distance (molecular area at least 3 nm in diameter), but which 



170 Mineralogical Analysis 

presents a degree of order at short distance that confers specific 
properties. The short-range arrangement takes into account the mutual 
arrangement with the closest neighbouring atoms at the scale of 
interatomic distance. These substances do not have a clear spectral 
signature in X-ray diffraction. With another medium or at the long- 
distance scale, different types of non-crystallinity can also be observed: 

- Zones that present substitution disorders or structural dislocation: this is 
the case of amorphous substances in a well-arranged periodic structure 
(structure with defects); high-resolution phase contrast transmission 
microscopy and scanning transmission electron microscopy (STEM) in 
micro diffraction mode allow this type of arrangement to be detected 
and localised. 

- Extended zones without periodicity composed of clusters of randomly 
distributed particles with a short-distance arrangement; this is the case 
of gels of alumina, iron, silica, and some aluminosilicates (opaline, 
allophane-like, proto-allophane, allophane, proto-imogolite, gel-like, 
glass-like, vitric silica etc.). 

Some substances display the beginning of medium-distance organi- 
zation (Fig. 6.1) which results in broad lines in XRD 2 (e.g. imogolite, 
ferrihydrite, feroxihyte). Spectroscopic techniques can be used to study 
these minerals (see Fig. 6.2): 
-EXAF 2 techniques provide accurate information on the interatomic 

distances of the closest neighbours and on the organization of the first 

layer of coordinance, but little information on relations between the 

polyhedrons (medium-distance coordination). 

- XANES 2 techniques enable analysis of the sphere surrounding an atom, 
but a good knowledge of the structure is a precondition for success. 

-NMR 2 spectrometry is selective for chemical structures but not very 
sensitive; the study of the hyperfine magnetic field of iron oxides 
makes it possible to measure the degree of crystallinity; in silica gels, 
the 29 Si nucleus enables the different states of Si0 4 bonds to be 
distinguished. Different forms of 31 P can also be studied. 

- ESR 2 and ENDOR 2 spectrometry enable analysis of the hyperfine and 
super-hyper-fine structures by electron spin resonance at the atomic 
scale. 



2 See abbreviations p. 168. ENDOR Electron nuclear double resonance. 



Selective Dissolution 



171 




Long-distance 
order v 



Short-distance 
order 



Expanded 
X-ray bands 



) 



J Crystalline 



Examples of short distance orders 



^-^ Micro-aggregate 

(SO) Water 
{Uyy^ monolayer 

AifiT Si0 2 /AI 2 3 =2 
Allophane 223^ 

sphere 

Si0 2 /Al 2 3 < 1 
(Okada1975) 

Allophane 
XRD spectrum 



compounds 

Allophane 2Si0 2 , Al 2 31 nH 2 
(hollow sphere structure s 35 A) 





21. 4A 



Imogolite 
XRD spectrum 



(Cradwicketal. 1972) 




Fig. 6.1. Crystalline and amorphous to X-ray compounds 



172 



Mineralogical Analysis 



All these instrumental methods use radiations that correspond to ranges 
of distance suitable for the sub-micronic scales needed for the study of 
the structure of soil materials. The range of radiation extends from radio 
frequencies to X-rays and gamma radiations. Their high purchase price, 
the degree of specialization of the equipment and their use in very 
specialised analytical fields limits these types of studies to highly 
specialised laboratories. 



Radio micro 

frequencies waves 



IR 



M IN 



~~ l~~ — ' — ^ 
10 5 10 



uv 



X-rays 



r-rays 



~i 1 1 r. 

10 1 10 _1 1C 



10 9 



10 7 



~i 1 1 1 1 1 1 1 1 1 1 1 r~ 

1Q 8 1Q 10 1Q 12 1Q 14 1Q 16 1Q 18 10 2 



nm 



V Hz 



i io i6o i'o 3 ' io 5 ' ib 7 ~~ ~76 9 



n cm 



w Sp[p transi tions 



(Nucleus) (Electroiji) 

M Molecular 



stations 



, ^/lolec jlfi 



vibratiDns 



x ternal ele ctrons 



Internal electrons 



Nucleus 



Nature of 
transitions 



Raman 



NMR 



ESR 



IR 



Mossbauer 



XRF 



XAFS 



Microwaves 



Techniques 



XANES EXAFS 



10" 



10" 



10" 2 



10 



10 4 



10 6 



Energy EV 



Fig. 6.2. Analyses of molecular structures using spectroscopic techniques (elec- 
tromagnetic spectra and environmental probes; A, wavelength; v, fre- 
quency; n, wavenumber; radiations: F, far IR; M, medium IR; N, near IR, 
V, visible; UV, ultraviolet); NMR, nuclear magnetic resonance; ESR, 
electron spin resonance; XRF, X-ray fluorescence; XAFS, X-ray 
absorption fine-structure spectroscopy; XANES, X-ray absorption near 
edge structure; EXAFS, extended X-ray absorption fine structure 



Instrumental methods allow observations at the sub-micronic scale, but 
chemical analysis enables analysis of entities that represent the average 
activity of certain particles because of their surface and their charge. Thus 
the continuum produced during weathering can be split in a satisfactory 
way by a series of extractions that make it possible to isolate compounds 
of increasing crystallinity that correspond, or not, to different chemical 
species. 



6.1.3 Selective Dissolution Methods 



The range of "selective dissolution methods" is obviously limited because 
of the diversity of soil minerals (Table 6.1) and the difficulty involved in 
dissolving a well-defined single phase (Table 6.2). 



Selective Dissolution 



173 



Dissolution depends on different factors: 
The size of the "crystal" and the level of atomic disorder 
Defects in stoechiometry or the blocking of active sites 
The properties of the crystallographic faces (anisotropy) 
The porosity of the systems, the density of surface defects, etc. 



\ 



pH action 
Complexing action 
Redox action 
Ionic concentration 
Sample:reagent ratio 



Sample 
matrix 



Nature, origin 

particle size, dispersion 

Crystallinity, reactive surfaces 

Temperature 



Chemical actions of 
extraction reagent 



| Solid residual phase 



X-ray Diffraction 
Thermal Differential Analysis 
Thermo Gravimetric Analysis 
Magnetic fractionations 
Mossbauer spectrometry 



Physical factors 
Experimental conditions 
Thermodynamics 



Soluble phase 



Reaction kinetics 

Equilibrium constants 

Chemisorption, 

Plan of contact 

Zeta potential function 

Agitation conditions 

Photolysis 



Weak flow 

(or closed system) 

weak solid:reagent ratio 



Outpu t balan ce | 

High flow 

(or open system) 

high solid:reagent ratio 



non-stochiometric 
dissolution 



Ion accumulation in solution 
risk of precipitations 
(complexing agent) 
delay factors 
oversatu ration 
inhibition of dissolution 



Precipitation extracted phase 
nucleation, reactional barriers 
phase recombinations: 
neooformations and 
matrix transformations 



Congruent, incongruent 
or selective dissolution 
stochiometric unloading 
at reaction interface 



Quick dissolution to 
equilibrium with 
residual matrix 



Fig. 6.3. Diagram of factors that control selective dissolution 



The reagents used and possible pretreatments should not cause 
precipitation. They should ensure the maintenance in solution of the 
extracted products and if possible, limit recombinations in the liquid 
phase (Figs. 6.3 and 6.4). 

Oxides, hydroxides, and oxyhydroxydes cause dependent charges in 
the soil and their structures, bonds, surfaces, and reactivities vary with 
their degree of crystallinity and the degree of disorder of their lattices 
(Table 6.1). 



174 



Mineralogical Analysis 



OH 



Nucleation 
Ageing Recombination 



Ions in 
solution 


► 


Hydrogels 


— ► 


Xerogels 




Crystalline 
forms 










* Syneresis - 







10" 2 M _ 



10" 3 Mh 
10" 4 M 




14 pH 



Fig. 6.4. Solubility of hydroxides as a function of pH and concentration (lower 
parts) and transformation of hydroxide gels (upper part) 



6.1.4 Reagents and Synthetic Standards 

The complexity of iron forms and of non-crystalline products often 
requires the use of pure synthetic models of minerals with a crystallinity 
or a short-distance atomic arrangement that closely resembles the 
substances found in the soil. 

The precipitates should be prepared starting from products with a high 
degree of purity, as hydroxides tend to adsorb impurities because of their 
very great specific surface. Flocculation is achieved by adding H + or OH" 
ions. Boiling causes transformation by dehydration and ensures the 
growth of the gel (nucleation). The time factor allows ageing of the gel, 
i.e. progressive slow crystallization (Fig. 6.4), transformation from a 
short-distance organization to an organization of a higher nature. For 



Selective Dissolution 175 

example, with aluminium in a ionic state, first precipitation of a monomer 
will be observed and then hydroxide: 

Al 3+ + 3 OH " -> Al(OH) 3 

in different steps: 

Al(OH) 2+ ^ Al(OH) 2 + -> Al 2 (OH) 2 4+ etc. 

dehydratation is accompanied by loss of H + during precipitation. With 
amphoteric aluminium compounds, the precipitates can be redissolved in 
alkaline medium forming soluble aluminates. All procedures must be 
strictly respected in order to obtain precipitation products that correspond 
to reproducible stages of formation. 

These procedures were defined by Henry (1958), Towe and Bradley 
(1967), Atkinson et al. (1968), Schwertmann and Taylor (1972; 1977), 
Murphy et al. (1976), Jeanroy (1983), Farmer and Fraser (1978), Pollard 
(1992), Lewis and Schwertmann (1979). 

Preparation of Iron Compounds 

Goethite 

- Dissolve 8.08 g of ferric nitrate (Fe(N0 3 ) 3 ,9H 2 0) in 80 mL water in a 
250 mL Erlenmeyer flask. 

- Bring the pH to 7.5 by adding approximately 20 mL of 3 mol (KOH) L" 1 
solution drop by drop (while on a magnetic stirrer). 

- Leave for 3 h to form a deposit, siphon the supernatant and wash the 
precipitate four times with water to eliminate any soluble potassium 
nitrate that has formed. 

- Suspend in 100 mL water, then add 3 mol (KOH) L" 1 solution to bring 
to a 0.3 mol (OH) L" 1 solution. 

- Store the solution in a polypropylene bottle at 20°C with occasional 
agitation for 2-5 years depending on the degree of nucleation desired. 

- Wash until elimination of KOH. 

- Dry in a ventilated drying oven at 50°C. 

Akaganeite 

- Grind a sample of pure FeCl 2 ,4H 2 in an agate mortar to pass through a 
0.5 mm sieve. 

- Spread in a thin layer and allow hydrolysis to occur in contact with 
humid air for 6 months (the product will turn brown over time). 

- Wash with H 2 to eliminate remaining Fe 2+ , then dry at 50°C. 



176 



Mineralogical Analysis 



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177 









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178 Mineralogical Analysis 

Lepidocrocite 

-Dissolve 0.6 g of FeCl 2 ,4H 2 in 150 mL of a 0.2 mol (NaCl) L 1 

solution saturated with nitrogen by bubbling using a peristaltic pump 

regulated at a flow rate of 15 mL min 1 . 

- Agitate under nitrogen and adjust pH to 6.0 by adding NaOH 1 mol L" 1 
drop by drop. 

-Allow the pH to stabilize, then replace nitrogen bubbling by air; 
maintain the pH during oxidation (2 h 30 min). 

- Wash with water, then dry at 50°C. 

Poorly ordered Ferrihydrite 

- Dissolve 2.02 g of Fe(N0 3 ) 3 ,9H 2 in 500 mL distilled water and bring 
the pH to 7.5 by adding 15 mL of 1 mol (NaOH) L" 1 solution drop by 
drop. 

- Store for 18 h at pH 7.5. 

- Wash with water and dry at 50°C. 

Hematite 

- Prepare a solution M of ferric chloride (FeCl 3 ,6H 2 0). 

- Precipitate with NH 4 OH at 60°C. 

- Filter on rapid filter. 

- Wash until the CI" test is negative (AgN0 3 test). 

- Calcinate for 1 h at 500°C. 

Maghemite 

- Weigh 5 g of ferrous oxalate (FeC 2 4 ,2H 2 0) and place it in a quartz 
crucible with a lid. 

- Heat gradually in an electric furnace at 410-420°C and maintain at this 
temperature for 1 h to eliminate water of constitution. 

The resulting product is close to maghemite. 

Organic amorphous iron 

- Extract the organic matter (OM) of a 20 g sample of podzolic soil with 
high humus content with 900 mL of 0.2 mol (NaOH) L" 1 solution. 

- Centrifuge the extract, then filter (calculate the OM content expressed 
as C content). 

- Prepare a ferric nitrate solution (Fe(NO 3 ) 3 ,9H 2 0) containing 108 g L" 1 . 

- In 225 mL of this solution (3.35 g of iron) add the desired proportion of 
OM extract. 

- Agitate while checking the pH with a pH meter; the final pH should be 
5.0; a brown-red gel will precipitate. 

- Wash with distilled water until complete elimination of sodium. 

- Preserve the gel in water and check the iron content (mg mL" 1 ). 



Selective Dissolution 



179 



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180 Mineralogical Analysis 



Preparation ofAI 3+ Compounds 

Boehmite 

- Prepare a 0.1 M solution of A1C1 3 . 

- Neutralize to pH 6 with 0.4 mol (NaHC0 3 ) L" 1 solution. 

- Leave in contact for one hour. 

- Bring to pH 8 by slowly adding 0.15 mol (NaOH) L" 1 solution. 

- Store in a closed plastic (PTFE) bottle for 60 h at 160°C. 

- Cool, wash until elimination of bicarbonate and remove surplus sodium 
hydroxy de by centrifugation. 

- Store in suspension in closed polythene bottle. 

Preparation of mixed compounds 

Imogolite 

- Place 30 mmol of aluminium perchlorate in 2.5 L of deionised water. 
-Add 15 mmol of tetraethyl silicate, this corresponds to approximately 

3.3 mL of commercial solution. 

- Homogenize, then bring the pH to 4.5 with soda. 

- The mixture will become opalescent; leave to stand overnight and the 
liquid will become clear. 

- Boil gently at reflux boiling point for 5 days. 

- Leave to cool, then add ammonia to gradually reach pH 9.0; the gel will 
precipitate. 

- Wash until elimination of sodium; store in water and measure the Al 
and Si concentrations. 



6.2 Main Selective Dissolution Methods 



6.2.1 Acid Oxalate Method Under Darkness (AOD) 



Principle 

This dissolution reagent is also called Tamm reagent. The method allows 
allophane and gels, iron, and aluminium organic complexes, hydrated 
oxides of iron, and aluminium (ferrihydrite, feroxyhite) to be dissolved. 
Imogolite is not completely dissolved in only one treatment. 
Phyllosilicates are only very slightly attacked, except if their level of 



Selective Dissolution 



181 



disorder is significant. Lepidocrocite is sensitive to oxalate reagent. If 
several treatments (2-3) are performed; some Al and Fe crystalline 
compounds can be solubilized to a considerable extent. 

The ammonium oxalate-oxalic acid buffer induces processes of 
protonation, complexation, and reduction. In this way it can cause the 
transfer of protons, electrons and ions (Stum 1985; Furrer 1985-1987; 
Cornell and Schindler 1987; Schwertmann 1991). 

The oxalate ion forms three complexes with ferric iron: 



Fe 3+ + C 2 6\- 


<=> 


Fe C 2 0} 


(6.1) 


Fe 3+ +2C 2 04 _ 


<=> 


Fe (C 2 4 ) 2 


(6.2) 


Fe 3+ +3C 2 C>4" 


<=> 


Fe(C 2 4 ) 3 " 


(6.3) 



(ferrous iron also gives complexes with other constants of stability and 
solubility). 

An excess of oxalate buffer is required to bring the equilibrium 
reactions to stage (6.3); if not the C 2 4 2 ~ acceptors H + and Fe 3+ can induce 
competitive reactions, particularly if raising the pH decreases the 
constants of solubility. 

With Al 3+ a complexation of the following type occurs: 



/OH _ OOC 

AK + 



OH 



.Al' 



-ooc 



c o 



c = o 



Below a pH of 3.5, the surfaces of the oxides are saturated with 
protons. The pH 3.0 zone is thus favourable because it controls charges 
below the point of zero charge (cf. Chap. 20). With respect to the 
reactional stages, protonation should be the first stage of dissolution of 
the compound, as this reaction allows better adsorption of the complexes 
and sumultaneous synergic action. 

In the case of non-crystalline compounds of iron and manganese, 
reduction is preponderant: 



Fe 3+ + e" 



^ Fe 



2+ 



182 Mineralogical Analysis 

The complexing reagent has two effects: 

- Ferric ions become less oxidizing as the ferrous ions are more reducing; 
the Fe 3+ :Fe 2+ ratio decreases, solubility increases; the reaction is 
autocatalytic 

- It affects pH (and maintains it at 3.0 with the buffer system) and thus 
influences the dissolution and stability of complexes; it prevents 
variations in the oxydoreduction potential which would otherwise be 
caused by the variations in pH. 

Changes in the Method 

Tamm (1922, 1931, 1934a,b) recommended an ammonium oxalate-oxalic 
acid buffer reagent to dissolve the inorganic gels including iron oxides, 
free silica, and alumina. This author suggested a pH of 3.25. 

A rapid examination of the composition of the reagents used since 
1922 in the Tamm method highlights the many different procedures used 
(e.g. variations in the concentration of the reagents, in pH, in the 
soil/reagent ratio, contact time, agitation, temperature, photolysis, see 
Fig. 6.5). 

Some changes were made to adapt the method to the nature of the 
sample and its components. Comparisons with old data are often difficult 
because of complexity of the soil matrix and interactions, and especially 
because the exact operating conditions are unknown. 

Jung (1934) stated that the method was appropriate for light soils, but 
not for calcareous soils, and that it did not give repeatable results with 
heavy soils because of the attack of clays. Many studies have been 
published on different soil types using varying concentrations of reagents 
and a pH ranging from 3 to 6, or using other organic acids (tartric, citric, 
salicylic, benzoic, phtalic, malonic acid, etc.) combined or not with 
reduction with H 2 S, nascent hydrogen, or dithionite (Duchaufour and 
Souchier 1966). 

A significant stage in the development of the Tamm method was the 
discovery of photosensitivity during dissolution (Schoefield 1959) and of 
the effects of photosensitization (De Endredy 1963, this method being 
known as Tamm-UV), and especially the establishment of a reference 
procedure by Schwertmann (1964) also called Tamm reagent in darkness. 
This method is now used as an international standard. 

The time factor has a random influence on the reactions, the 
dissolution process generally being rapid at the beginning but tending to 
slow down considerably after 4 or 5 h. The influence of time is generally 
only critical if it is less than 2 h. Schwertmann fixed the average time at 
2 h on the basis of a profile of traditional dissolution indicating the 
preferential dissolution of certain fractions (ferrihydrite, substances with 



Selective Dissolution 



183 



short-distance arrangement, etc.). Mc Keague and Day (1966) showed 
that in their conditions, dissolution was better when the contact time was 
increased to 4 h (in darkness). Although a sequence of treatment of this 
duration can have a kinetic effect, only one extraction is the basis of this 
method. 



Tamm1922 

pH 3.25 

1 :50 ratio 

2h 

light radiation 



Random results 



Active iron + 
crystallised 

Tamm UV 



Schofield1969 
Deb1950 



"Tamm darkness 



De Endredy1963 

pH 3.25 r= 1/100 0.2 M 

1h under UV 



Schwertmann 1964 
pH3r= 1/100 0.2 M 
2 h darkness 



I 



Active non-crystalline 
or cry ptocrystal line 
iron 



International standards 
USDA 1972(4h) 
Blakemore 1981 (4h) 
Jackson 1982 (2h) 
Jeanroy 1983 (4h) 
ISRIC1987(4h) 
Wilson 1 987(4 h) 



McKeagueand 
Day (1966) 
pH3 r=1/40 
0.2 M4h 



pH 3.5 0.15 M 
darkness 
Higashi 1974 
lnoue1981 
Parfitt1982 



Fig. 6.5. Changes of the Tamm method using ammonium oxalate-oxalic acid 
reagent (basic procedures) 



The soil/solution ratio generally has a limited influence on the results. 
Parfitt (1989) showed that extraction using the 0.15 M reagent at pH 3.0 
with a soil/solution ratio of 1:100, agitation for 4 h at 20°C (in darkness) 
was satisfactory for many soils, but cannot be used if Al or extractable Fe 
exceeds 5%. In this case it is necessary to use a 0.20 M reagent and a 
ratio of 1:200. 

Extraction with 0.15 M oxalate or 0.20 M reagents at pH 3.0 gives 
equivalent results for allophane if the concentration of allophane does not 
introduce a limiting factor. 

The pH of extraction is critical. It controls the kinetics of dissolution of 
the crystalline and non-crystalline compounds. Maximum dissolution is 
reached at a pH of between 2.6 and 3.0, the protonation being synergistic 
with the reducing and chelating action of the oxalate reagent. If the pH 



184 Mineralogical Analysis 

rises above 4.0, the effectiveness of the buffer decreases drastically, the 
extracted quantities of iron decrease and selectivity is modified. The pH 
should thus be fixed at 3.0, to prevent possible variations. Temperature 
accelerates the reaction and modifies selectivity. The standard tempera- 
ture is around 20°C. 

Preparation of the Reagents 

All the reagents should be prepared with reference products, bi-distilled 
water, or water deionised on a resin column that can fix Si. 

Acid ammonium oxalate: 0.2 M oxalate-pH 3.0 Tamm reagent 
-Dissolve 16.15 g of ammonium oxalate (COONH 4 ) 2 ,H 2 3 and 10.90 g 

of oxalic acid (COOH) 2 ,2H 2 4 in approximately 900 mL of water; 

complete to 1000 mL. 
-Check the pH and bring it to pH 3.0 by adding ammonia or 0.2 M 

oxalic acid. 

- Prepare each week and store in a brown bottle protected from the light. 

- 0.2% superfloc (flocculation agent) in water (Cyanamid Corp.). 

- Matrix corrector for dilutions before atomic spectrometry: for 10,000 
ppmK 

weighl9gofKCl, 

dissolve in approximately 900 mL of water, 

when the temperature of the solution reaches ambient temperature, 

complete to 1,000 mL. 

Procedure 

-Measure soil moisture on a separate sub-sample to determine the 
moisture correction factor (cf. Chap. 1). 

- On the laboratory balance, weigh 1 g of air-dried soil sieved with a 0.2 
mm mesh (avoid over- grinding). 

-Put in a 100 mL bottle. 

- Add 50 mL of acid oxalate reagent; for soils with high extractable- 
oxalate compounds (extracted Al or Fe compounds >2%) add 100 mL 
oxalate reagent and use a 250 mL bottle. 



(NH 4 )2C204,H 2 0, mw: 142.12; can be awkward for certain clays (and can be 

replaced by sodium oxalate Na 2 C 2 4 ); safety: a classified poison, do not 

ingest. 
COOH-COOH, 2H 2 0, mw: 126.07 decomposed by UV radiation; drying at 

100°C involves losses by sublimation, decomposition at 160°C; safety: a 

classified poison - caustic - do not ingest). 



Selective Dissolution 185 

- Agitate 4 h in darkness. 

- Decant part of the supernatant in a 50 mL centrifugation tube. 

- Centrifuge for 10 min at 10,000 g; if the liquid is not perfectly limpid, 
resuspend, add 3 drops of superfloc and centrifuge again (two reactive 
blanks should be integrated into each series in addition to a soil 
standard of reference and two replicates on a sample of the series). 

After adequate dilution, analyses are carried out on this extract: 

- Si: ICP or AAS spectrometry at 25 1 .6 nm, N 2 0/C 2 H 2 flame 

- Fe: ICP or AAS spectrometry at 248.3 nm, air/C 2 H 2 flame 

- Al: ICP or AAS at 309.3 nm, N 2 0/C 2 H 2 flame 

- Mn: ICP or AAS at 279.5 nm, air/C 2 H 2 flame 
and if necessary, Ti and P. 

If absorption spectrophotometry is required: 

- Destroy the oxalate matrix by boiling with concentrated nitric acid 

- Bring to dry and dissolve in 5 M hydrochloric acid 

-Evaporate to almost dry and dissolve in water, then complete to the 
required volume 

Calculations 

Data collected 

- Pj, weight of wet or air-dried soil sample for measurement of moisture. 

- P 2 , weight of the soil sample dried at 105°C. 

- P, weight in mg of soil sample for extraction. 

-A, £, contents in the extract and the blank respectively (mg L" 1 ). 
-D, dilution of the extract. 

- V R , mL of the oxalate reagent used for extraction. 

Moisture correction factor 

This measurement is essential to bring back the results to soils dried at 
105°C, especially for all soils rich in non-crystalline substances like 
allophanic soils: 

# = 100 ^— —(%) 
P x 

Moisture correction factor =/= 100/// 

Calculation of contents of elements (Fe, Al, Si, Mn, Ti, P, etc.): 
% element = 0.1 (D V R f(A-B))/P 

Conversion factor of the content of an element to oxide content: 
% Fe 2 3 = % Fe x 1 .43 % Mn0 2 = % Mn x 1 .58 

% A1 2 3 = % Al x 1.89 % Si0 2 = % Si x 2. 14 



186 Mineralogical Analysis 

Remarks 

The extraction is reliable enough for most soils. Identification of certain 
phases may sometimes be difficult when clays are disordered. Even when 
extracts are protected from the light, they can still undergo change. They 
should thus be analysed rapidly to avoid precipitations due to the 
instability of the reagent. 

The addition of superfloc is generally not necessary given the strong 
ionic force of the reagent and the resulting complexes. The supernatant 
liquid can be filtered by siphoning with a syringe equipped with a 0.45 
nm filter (after decantation). 

Oxalate extraction is used in a number of different fields. 

- Pedology and pedogenesis, geochemistry (differential dissolutions and 
identification of the non-crystalline or little ordered phases, transitions 
between crystalline phases, chemical and methodological studies, effect 
on the soil structure, etc.) preferably using data from the dissolved 
phases. 

- Mineralogy uses the solubilized phases and the residual solid phases 
simultaneously for: 

- The study of substitutions, order-disorder states, compounds 
with short-range atomic arrangement; 

- For the preparation of samples (dissolution of cements between 
the particles, elimination of oxides to improve the intensity of 
diffraction of the crystalline compounds), to carry out differential 
XRD analyses (DXRD) and to enable analysis after elimination 
of paramagnetic compounds (Mossbauer, ESR, EXAFS 
spectrometries), to observe spatial distribution on thin sections of 
the soluble oxalate phases (Arocena 1988), and finally to model 
deterioration processes (resulting compounds, etc.). 

-Microbiology and agronomy to analyse biophysical and biochemical 
activity (effect on water retention, plasticity, availability for plants of 
active iron linked to oxalic acid contents generated in the soil (oxalic 
acid of biochemical origin causes the disruption of iron oxides). 

- In the case of calcareous soils, ammonium oxalate precipitates Ca ++ 
cations in the form of calcium oxalate with solubility lower than 0.006 
g per litre of water or acetic acid; the carbonate must thus be destroyed 
with the minimum quantity of acetic acid necessary before extracting 
ammonium oxalate and complementary measurements of the elements 
solubilized with acetic acid. 

-In reducing conditions (e.g. hydromorphic soils, histosols, andosols 
under permanent wet climate), the oxalate method cannot provide 



Selective Dissolution 187 

information on the initial state of oxidation of iron and manganese in 
the soil before extraction. 
- In andic soils and andosols-andisols the oxalate method can extract as 
much as the DCB method (cf Sect. 6.2.2); the organic complexes of 
iron are dissolved; allophane, which is extracted after 2-4 h agitation 
with oxalate, can be estimated using the values for extracted silicon 
based on the hypothesis of the prevalence of Si-O-Al bonds; 
ferrihydrite can be estimated starting from extracted iron. 



6.2.2 Dithionite-Citrate-Bicarbonate Method (DCB) 

Principle 

This method (Mehra- Jackson 1959-60) makes it possible to solubilize 
pedogenic oxides and hydroxides: 

- crystalline iron oxides (hematite, goethite), non-crystalline iron oxides 
and iron and aluminium organic complexes, as well as exchangeable 
iron and manganese oxides, some non-crystalline compounds with a 
Si0 2 :Al 2 3 ratio of less than 0.5; 

- magnetite and ilmenite are only slightly attacked, as are gibbsite and 
allophane-imogolite aluminosilicates; however, magnetite can be 
significantly solubilized in certain cases (magnetite is strongly oxidized 
into maghemite in very oxygenated medium); 

- clays are not affected, but any iron present in the lattice of vermiculites 
and non-tronite can be significantly solubilized, particularly if the 
extraction pH is lowered; 

Reduction is the predominant process of this method, dithionite being a 
very active reducer (Deb 1950) below pH 9-10. Biologically reducible 
elements like iron or manganese are reduced and maintained in solution 
by complexation with the citric acid in the system buffered at pH 7.3 with 
sodium bicarbonate. 

The optimum pH for reduction is 7-8. Below pH 6.5, colloidal 
sulphur can precipitate resulting in a suspension in the extracts that 
prevents measurement by absorption spectrometry. The use of buffered 
medium limits this phenomenon. As the dithionite solution rapidly 
loses its reduction properties, complexation avoids reoxidation as well 
as the precipitation of iron sulphide and allows maintenance in solution 
of the extracted phases as long as the extraction time does not exceed 
15 min. 

Initially, only one addition of dithionite was performed on the first 
extract. Subsequently, as a result of international influences and on the 



188 Mineralogical Analysis 

recommendation of the initiator of the method, two additions of dithionite 
were recommended. 

If required, two or three successive extractions can be performed to 
include crystallized iron compounds of relatively significant size. In this 
case, the extracts can either be mixed before analysis or analysed 
individually to measure a kinetic evolution of the solubility, summation is 
only carried out after each measurement. 

The temperature should be set at 75 °C to accelerate the reaction and to 
limit the appearance of colloidal sulphur and iron sulphide, but also to 
minimize dithionite decomposition. This temperature should not be 
exceeded, and localised overheating should be avoided by using a water- 
bath. 

The iron contents should not exceed 0.5 g Fe 2 3 in order to obtain an 
excess of reducer and complexant. The reaction of iron reduction in a 
slightly basic medium can be written: 

S 2 4 2 " + 4 OH" -> 2 S0 3 2 " + 2 H 2 + 2 e" 

2 Fe 3+ + 2 e" -> 2 Fe 2+ 
and in citric complexing solution 

S 2 4 2 " + Fe 2 3 + 2 HOC(COO) 3 3 - + 2 H + -> 2 S0 3 2 " + 2 Fe 11 - 
HOC(COO) 3 ~+H 2 

The weight of the sample must be between 1 and 5 g without 
modifying the composition of the reagent (if iron cannot be complexed 
due to insufficient citrate, precipitation of black iron sulphide may occur). 

A buffered medium is used to avoid a change in pH resulting in 
variations in the Redox potential, each Fe 3+ requiring two OH" during 
reduction. 

Certain authors tested reduction methods using dithionite in citrate 
with variable pH (Homgren 1967; Avery and Bascom 1982) or Tamm 
reagent (Duchaufour and Souchier 1966; Hetier and Jeanroy 1973; 
Loveland and Bullock 1976) or in other buffered and complexing 
mediums such as sodium tartrate-acetate (Deb 1950) or in a medium with 
a basic pH such as pyrophosphate (Franzmeier et al. 1965). 

Complexes with citric acid (a tridentate sequestering agent) are similar 
to those formed with oxalic acid and give very stable iron and aluminium 
compounds. Aluminium citric acid complexes have a stability constant, 
log Kl = 7.37. 

In the natural environment, the presence of citrate prevents, or delays, 
the precipitation of aluminium, as the sites of coordination are occupied 
by citrate, its hydrolysis is slowed down: 



Selective Dissolution 189 

\l ^ 
Al< >AP 



\K x At^(XX-0 

1 x o o— 9^9— CH 2 COO 

O CH 2 

Replacement of the water molecules and blocking of the sites of 
coordination occurs in the DCB extraction medium, which has a high 
concentration of citric acid. Hydrolysis thus becomes impossible (Kwong 
and Huang 1979). Some minerals, for example pseudo-boehmite, present 
a particular affinity (Cambier and Sposito 1991). 

Two other reagents (hydroxylamine hydrochloride and acidified 
hydrogen peroxide) were found more efficient than DCB method for 
selective dissolution of manganese oxides (Neaman et al. 2004a,b). 

The DCB treatment can cause structural disorders which can be 
observed by XRD or electron micro-diffraction. The adsorption of citric 
groups on certain clays can considerably slow down the departure of 
interfolayer water. This should be taken into account in the analysis of 
the residue containing the forms known as "free". It should be noted that 
under these conditions, proto-imogolite cannot be transformed into the 
better structured imogolite. 

Preparation of the Reagents 

All the reagents should be reference products for analysis, water should 
be bidistilled or possibly deionized on resins suitable for the elimination 
of silica. 

- Sodium dithionite in powder form depending on the number of 
analyses, only small bottles of the product should be used in order to 
always have fresh product available. 

- Citrate-bicarbonate buffer: dissolve in distilled water before use 79.40 g 
of trisodium citrate (C 6 H 5 Na307,2H 2 0), 9.24 grams of sodium bi- 
carbonate (NaHC0 3 ), check the pH which should be 7.3 approximately, 
bring to 1 L. 

- Flocculation use either 400 g saturated sodium chloride, NaCl in 1 L of 
water, or 375 g saturated potassium chloride, KC1 in 1 L of water, if 
further measurements by atomic absorption or ICP spectrometry are 
required, potassium chloride should be used in order to avoid stronger 
sodium concentrations, acetone. 



190 Mineralogical Analysis 

Procedure 

-In a flat-bottomed 100 mL centrifugation tube made of polypropylene 
or PTFE, place from 1 to 5 g of soil (0.2 mm particle size ) depending 
on the estimated concentration of ferric oxide (carbonates, organic 
matter and soluble salts must be removed from the sample beforehand). 

- Add a bar magnet and 45 mL of buffered citrate -bicarbonate reagent by 
means of a fraction distributor equipped with a PTFE syringe. 

- Place on a immersed magnetic stirrer in a water bath regulated at 75°C; 
when the sample reaches the temperature of the bath, using a measure, 
add 1 g of dithionite powder and continue to agitate at moderate speed 
for 5 min. 

- Add another gram of dithionite and agitate for 10 min. 

-After 15 min digestion, centrifuge for 5 min at 2,500 g to obtain a 
limpid solution (if the liquid is still cloudy, suspend and add a saturated 
solution of sodium or potassium chloride to cause flocculation then 
centrifuge again at 2,500 g; this treatment will make the centrifugation 
pellet more compact and thus complicate resuspension for a 2nd 
treatment; for soils originating from volcanic ash, it is often necessary 
to add 10 mL acetone before centrifugation to achieve satisfactory 
flocculation). 

- Decant the clear supernatant liquid in a 250 mL volumetric flask. 

- If the residue displays intense brown, black, or red colour, add 45 mL 
of buffered reagent and treat as above with two additions of dithionite 
and heat for 15 min at 75°C, re-suspend the compact centrifugation 
pellet to allow a homogeneous attack. 

- Centrifuge and decant in the same 250 mL flask (or analyze the second 
extract separately). 

-Wash the residue two or three times with 10 mL of buffered reagent, 
flocculate, centrifuge the rinsing products and add them to the previous 
extract. 

- Add 250 mL distilled water and homogenize. 

In each series, introduce two blanks (reagents only) and a reference 
sample. After adequate dilution (2-10 times) the filtrate containing free 
oxides and hydroxides should be analysed by atomic absorption 
spectrometry: 

- Al at 309.3 nm with an acetylene-nitrogen protoxide flame. 

- Fe at 248.3 nm with an acetylene-nitrogen protoxide flame. 

- Si at 251.6 nm with an acetylene-nitrogen protoxide flame. 

- Mn with 279.5 nm with an acetylene-air flame. 

- Ti, P, K, Mg can be also analysed if necessary. 



Selective Dissolution 191 

If colorimetry is used, certain methods make it possible to operate 
directly on the extracts, but it is preferable to destroy the buffered, 
chelating and reducing matrix by boiling with nitric or sulphuric acid and 
perhydrol. Iron is measured using 1-10 orthophenantrolin or ferron, 
aluminium using eryochrome cyanin, silica using molybdate taking 
phosphorous into account (cf. Chap. 31). 

Weigh the purified residue. The residue can be analysed using XRD, 
an instrumental method; the intensity of the lines is improved by DCB 
treatment (cf. Chap. 4); IR, ESR, NMR, EXAFS spectrometry (cf. Chap. 
12); thermal analysis, DTA-TGA (cf. Chap. 7); or chemical analysis 
(total analysis, cf. Chap. 31); CEC (cf. Chap. 26); dissolution of 
aluminosilicates, etc.). 

Calculations 

Data collected 

-A, B: respective contents in the sample and blank extractions in mg L" 1 . 

- D: dilution factor. 

- /: moisture correction factor (cf. "Calculations" under "Acid oxalate 
Method under Darkness"). 

- P: weight of air-dried sample in mg. 

Calculations 

Oxide percentages should be calculated for all the elements: Fe 2 3 , 

A1 2 3 , Si0 2 , etc. The "weight of the initial sample" minus the "weight of 

residue" enables total free oxides and hydroxides to be calculated: 

Al, Fe, Si... % = 25 (A-B) Df/P 

See "Calculations" under Sect. 6.2.1 for the conversion factors of elements 

into oxides. 

Remarks 

This method gives reasonably reproducible results if the crystalline iron 
forms are sufficiently fine to offer enough surface area to allow a 
significant attack. Many different procedures have been proposed, but the 
current standard method is identical for the reagent concentration to that 
initially suggested by Mehra and Jackson (1959): 0.42 g of sodium 
bicarbonate and 3.52 g of sodium citrate, 2H 2 in 45 mL of water. The 
main modifications one of the authors made of the method are: double 
reduction of dithionite on the same extract and the preparation of a single 
buffer-complexing reagent, which simplifies handling. 



192 Mineralogical Analysis 

The colour of the residue gives a good indication of the effectiveness 
of the treatment, but the presence of magnetite or ilmenite, which are not 
attacked by the DCB treatment, can colour the residue black or gray. The 
DCB method can be used to facilitate dispersion of clay whose 
suspension may be obstructed by pedogenic oxide and hydroxide 
coatings. 

The method of Holmgren (1967), whose reagent is composed of a 
rather unstable mixture of 17% sodium citrate and 1.7% sodium 
dithionite, is now sometimes used instead of the DCB method. It is 
considered to be equivalent to the Mehra- Jackson method, but simpler to 
implement and thus more suitable for repetitive analysis. 

Grinding to 0.2 mm allows a better attack of the iron forms present in 
concretions for example. Grinding is consequently generally performed 
to enable comparisons to be made, and in particular to compare the 
weight of the residues after treatment. 

Heating to temperatures above 80°C (or local overheatings) can cause 
the precipitation of black iron sulphide. In this case it is better to start the 
analysis again than to eliminate the precipitate with acetone and carbon 
tetrachloride. 

Dithionite treatment modifies the Fe 3+ :Fe 2+ ratio. 

Ryan and Gschwend (1991) suggested replacing dithionite with 
titanium III in the citric-bicarbonate + ethylene diamine tetraacetic acid 
(EDTA) solution. This reduction method, with the very complexing 
reagent at a temperature of 80°C, should theoretically enable more 
complete dissolution of the amorphous ferric oxides and goethite. But 
hematite is less solubilized. Extraction is more easily achieved with the 
Ti (III) method than with the DCB method, which gives the Ti (III) 
method a more selective spectrum of dissolution. The behaviour of the 
aluminium compounds is different. The use of the cold Ti (III) method 
increases the degree of selectivity, but the titanium content of the soil 
must be low. 

6.2.3 EDTA method 



Principle 

The EDTA (salt of Na) method according to Borggaard (1976), enables 
extraction of iron in an amorphous or very little ordered state as a result 
of biological deterioration (inorganic and organic non-crystalline iron) 
the ferrihydrite is dissolved. Both the water-extractable iron and the 
exchangeable iron must be in solution. 



Selective Dissolution 193 

This reagent is not suitable for the extraction of the amorphous and 
organic forms of aluminium because of the high pH of the extraction 
solution. The silicates and crystalline forms of iron and aluminium are 
not dissolved. 

The repeatability and selectivity of iron extraction are good. The 
method is reliable, but has one major disadvantage: the slowness of the 
extraction, the balance being achieved only after approximately three 
months of extraction. The most widely used process is hydrolysis and 
complexation in basic medium at ambient temperature (20°C). 

EDTA is an aminopolycarboxylic acid with six atoms suitable for the 
formation of chelates (from the Greek Khele = crab grip) to which a 
metal cation is linked by coordination with the organic radical (e.g. two 
atoms of nitrogen and four carboxyl groups, see Fig. 6.6). The six 
positions around the metal (Fe 2+ ) give a high complexing capacity and 
low selectivity, as most of the di- and trivalent elements are able to enter 
this type of complex. The constants of stability, log K at 20°C, are 13.8 
forMn 2+ , 14.3 for Fe 2+ , 16.1 for Al 3+ , 25.1 for Fe 3+ ). 

In an alkaline solution (pH 10), 1:1 complexes are formed with 
pedogenic elements. An excess of complexing reagent is needed for 
satisfactory control of the dissolution rate . 

Depending on the pH, it may be possible to obtain H 4 Y, H 3 Y", H 2 Y 2 , 
HY 3 " forms (Y 4 ~ being the EDTA anion, see Fig. 6.6). 

HOOC-CH 2X ^ CH 2 -COO 

H— N + — CH 2 - - CH 2 — NT- H 



OOC-CH/ ^CH 2 COOH 



H 2 C- 




Fig. 6.6. Molecule of EDTA IV (top) and coordination complex with iron (bottom) 



194 Mineralogical Analysis 

In the extraction continuum, the extractable forms of iron with EDTA 
appear to be linked with the most active forms in pedogenesis, i.e. with 
the "free" least crystalline compounds with extensive surface contact and 
thus great reactivity. Both exchangeable iron and organic chelated forms 
are solubilized because the pH of the reagent is high and the EDTA 
complexes are stable. 

In agronomy, the availability of iron for plants (nutritional factor or 
possible chlorosis) is often checked using also two other complexing 
reagents (Lindsay -Norvell 1976): diethylene triaminopentaacetic acid 
(DTP A) or ethylene diamine di(o-hydroxyphenylacetic acid (EDDHA). 

The correlation between EDTA- and oxalate-extractable iron is good: 
the ferrihydrite is dissolved by EDTA or by oxalate, but soils containing 
hydroxyferric complexes can react differently with these reagents 
(Jeanroy 1983). The time factor is significant. The extraction profile is 
slow and dissolution is continuous up to around 90 days, when it 
stabilizes. 

The temperature is critical and attempts carried out to accelerate the 
reactions by raising the temperature to 75°C, as in the DCB method, 
resulted in unacceptable displacement of selectivity, some crystalline 
products becoming attackable and solubilizable. 

The effect of pH was tested by Borggaard (1976). It cannot be below 
7.5. A pH of 10, similar to that used in the pyrophosphate and tetraborate 
methods, makes it possible to compare the organic phases extracted using 
the above methods and eliminates the effect of pH. But selectivity is 
random for aluminium, which, at a pH of 8-9, gives soluble aluminates. 

In EDTA, the concentration factor appears to have little influence on 
extraction, the kinetics of the reaction being controlled by hydrolysis. 
Intermittent agitation makes it possible to renew the reagent at the liquid- 
solid interfaces, thus avoiding the phenomena of local saturation. 
Clarification of the extracts is generally problem free. 

Preparation of the Reagents 

The initial procedure of Borggaard (1976) is a dynamic method. This 
author tested the effects of a concentration of the complexing reagent of 
between 0.01 and 0.1 M and a pH of between 7.5 and 10.5. This method 
is thus extremely long and cannot be adapted in its original form for 
repetitive analyses. 

A concentration of 0.1 M of EDTA at a pH of 10 is used as standard. 
This enables comparisons with the reagents which extract organomineral 
complexes at the same pH, i.e. pyrophosphate at pH 9.6-10 and 
tetraborate at pH 9.7. Commercially available EDTA is often in the form 
of sodium salt and is sold under different names: Versenate, Sequestrene, 



Selective Dissolution 195 

Titriplex II, Trilon B, etc. The empirical formula Ci Hi 6 N 2 O 8 corresponds 

to a molar mass of 292.25 g. 

Solution A: weigh approximately 29.225 g EDTA in 500 mL water. 

Soution B: dissolve 20 g NaOH in 250 mL water. 

Gradually mix B in A to bring the pH to 10. 

Complete to 1 L with distilled water. 

Procedure 

- On an analytical balance weigh precisely 2 g of soil ground to 0.2 mm 
in a 100 mL polypropylene or PTFE centrifuge tube. 

- Add 50 mL of 0. 1 M EDTA reagent. 

- Stop the tube and agitate with the Vortex vibrator for 1 min. 

- Place the samples on mobile plates that can be used on an oscillating 

agitator and store the series protected from the light for 90 days with 
daily agitation for 5 min. 

- After 90 days of contact, centrifuge at 5,000 g for 5 min and filter. 

The extracted elements (mostly Fe, Al, Si, and P) are analysed by 
plasma emission or atomic absorption spectrometry. When absorption 
spectrocolorimetry is used, the EDTA matrix has to be destroyed (cf. 
"Procedure" under "Acid Oxalate Method Under Darkness"). In each 
series, introduce two blanks with only reagents and a reference sample. 

Calculations 

Data collected: 

A, B, D, f 9 P of the same type as in the preceding methods (cf. 

"Calculations" under section 6.2.1 and "Calculations" under "Dithionite 

- Citrate - Bicarbonate Method (DCB)" in this chapter). 
Contents Fe, Al, Mn, Si, P (%) = 5 (A-B) DflP 

These contents are expressed as per cent of oxides (cf. "Calculations" 
under section 6.2.1). 

Remarks 

This method is one of the most reproducible and selective for amorphous 
iron and can also extract organometallic complexes if the pH exceeds 
nine. The extracted iron must be compared to the iron extracted by the 
oxalate reagent taking into account the fact that aluminous products 
dissolved at high pH can release iron for example resulting from 
isomorphic substitutions. 



196 Mineralogical Analysis 

It is useful to compare the results with the extraction of active iron 
available to plants (using DTPA or EDDHA reagents) to link the results 
to the phenomenon of chlorosis and to proceed from pedogenic 
observations to the identification of agronomic properties of the soil- 
plant-climate relationships. From an agronomic point of view, the 
extraction of phosphorous by EDTA is also useful to identify the 
proportions of P linked to Fe or Al which form part of the pool of 
"available P". 

6.2.4 Pyrophosphate Method 



Principle 

This method is used to analyze the forms of iron and aluminium 
complexed with the soil organic matter, in particular to differentiate the 
spodic and podzolic horizons where displacement of these complexes can 
be observed. 

Generally a good correlation can be obtained between extracted Al, Fe, 
and organic C. Well-crystallized iron oxides like goethite and hematite 
are not attacked and slightly ordered iron oxides are only slightly 
solubilized. 

The original procedure recommended by Alexandra va (1960), and 
given permanent form by McKeague (1967), cannot be applied as it 
stands but has to be modified with regard to the clarification of the 
extracts. 

The pyrophosphate anion P 2 7 4 has chelating properties and can react 
with polyvalent cations to give insoluble compounds and soluble 
complexes with organic matter, for example: 

R(COO) 4 Ca 2 + Na 4 P 2 7 -► R(COONa) 4 soluble + Ca 2 P 2 7 | 

But the complete mechanism of pyrophosphate action is not as clear as 
in the methods described earlier. The action of pyrophosphate has been 
questioned with regard to the organic forms of iron and aluminium: on 
one hand concerning the procedures and the reliability of the 
measurements, and on the other hand with regard to the mechanisms of 
extraction and the nature of the extracted products. Klamt (1985) 
mentioned "the denunciation of pyrophosphate extraction of Fe from 
soils (highly unreliable)", indicating that an international consensus had 
not been not reached. 



Selective Dissolution 197 

With regard to the procedures 

The time factor of 16 h is regarded as critical, particularly when the 
results of this method are compared with those obtained by EDTA 
extraction after a contact time of 90 days. 

The pH, which was tested at different levels, is here fixed at 10.0 to 
enable comparison with the other extraction methods in basic solutions. 

Pyrophosphate has chelating properties. Originally the choice between 
the use of sodium or potassium pyrophosphate was more or less random. 
K pyrophosphate extracts slightly more than Na pyrophosphate and 
makes spectrometric measurements easier by avoiding strong Na + 
concentrations, which are always awkward. But with certain clays, 
potassium has serious disadvantages 5 . Na pyrophosphate is consequently 
considered to give the best reproducible extraction and it is now used at a 
concentration of 0.1 M (Loveland and Digby 1984). 

At pH 10, pyrophosphate has peptizing properties which make the 
extracts very difficult to purify. Suspended particles are mainly 
responsible for the low rate of reproducibility and the lack of precision of 
the method, as the material in suspension is not chemically extractable by 
pyrophosphate. The efficiency of its centrifugation (speed and time of 
centrifugation) has been tested up to 100,000 g and compared to ultra- 
fitration. A flocculating agent must be added before centrifugation, 
usually sodium sulphate at concentrations ranging between 0.25 and 1 M 
(Schuppli et al. 1983) or superfloc cyanamid N-100 (or Floerger 
Kemflock F 20 H). In this case the concentration is critical (Ballantyne 
etal. 1980). It is fixed at 0.2 mL superfloc for 50 mL of extract. 
Centrifugation at 20,000 g for 15 min results in clear extracts. Ultra 
filtration with 0.02 |Lim millipore filters can be used to eliminate any 
colloidal particles that may still be in suspension. In this case 
reproducibility is about 10-15%. 

With regard to the mechanisms of solubilization 

In the reaction between sodium pyrophosphate and soil, complexation 
cannot be the main mechanism, as iron linked to complex organic forms 
cannot be dissolved without solubilization of the amorphous forms of 
iron which are very reactive, as in the case of EDTA solubilization. 



5 For example the K + ion is specifically adsorbed by vermiculite or deteriored 
micas because its diameter is compatible with the size of the adsorption sites. 
The selectivity of K + with respect to Ca 2+ or Mg 2+ is increased by the hydroxy 
aluminous polymer deposits on the interfoliaceous surfaces. In the presence 
of a strong concentration of P, and in certain conditions, K and aluminous 
oxides together can result in the formation of taranakite. 



198 Mineralogical Analysis 

Compared to amorphous iron oxides, the iron pyrophosphate complex is 
not considered to be very stable at pH 10. 

Bruckert (1979) considered that sodium pyrophosphate shifts the 
organic matter of its complexes of coordination with the metallic sites of 
clays (ferric bridges) and is adsorbed instead of the humic compounds 
which are solubilized at an alkaline pH. Metallic compounds with a high 
charge, such as amorphous hydroxides, can behave in a similar way. All 
the complexes extracted by pyrophosphate comprise the "immovable 
complexes". 

Micro-aggregates are destroyed and clayey and colloidal cements are 
dispersed. Fulvic acid-amorphous iron hydroxides complexes are 
extracted along with the organic molecules in the coatings on clays. 

Jeanroy (1981,1983) considered that dissolution induces a mechanism 
of peptization and solubilization. Adsorption of pyrophosphate on the soil 
particles increases the negative charges and increases their solublity in 
water. In contrast to EDTA, which has a flocculating effect, 
pyrophosphate puts ferruginous particles into suspension and these 
subsequently disperse in the extracts. 

Separation on millipore filter shows that in EDTA extracts iron is 
linked to small molecules and thus passes through the membrane. On the 
other hand, the pyrophosphate extracts contain compounds of greater 
molecular size that do not pass through the membrane, as only a small 
proportion of the chelated fraction is able to do so. Similarly 
ultracentrifugation of the EDTA extract does not separate the phases, 
clearly demonstrating that iron is in soluble chelate form, whereas 
pyrophosphate results in a significant colloidal centrifugation pellet. 

To summarize: with its peptizing action, pyrophosphate puts into 
suspension fine ferruginous particles, probably of ferric hydroxides, 
whose smoothness and small degree of atomic order are explained by the 
presence of organic matter which inhibits the crystallization of iron 
oxides. Bruckert 's "immovable complexes" appear to be mainly 
hydroxyferric complexes that reveal the preponderance of the mineral. 

From a practical point of view, pyrophosphate is effective only if the 
soil is under the influence of organic matter. In the opinion of Schuppli 
et al. (1983), the precipitation of iron in the pyrophosphate extracts could 
be due to the ageing of the extracts. Another dissolution mechanism 
could be the release by sodium pyrophosphate of small quantities of iron 
in the organic complexes, leaving these complexes negatively charged. 
They then become water soluble. The organic matter makes it possible to 
maintain quantities of iron in solution, but if the ratio reaches a certain 
level, precipitation will occur (Petersen 1976). 



Selective Dissolution 199 

If this description of the solubilization mechanism is accurate, 
pyrophosphate extraction could be a selective dissolution technique, 
especially if uncertainties concerning the purification of the extracts are 
overcome. From a practical point of view, pyrophosphate extraction 
enables the behaviour of certain soils to be differentiated for the purpose 
of classification, though without identifying the precise origin of the 
extracted iron; however organic forms are considered to be the most 
probable. 

Preparation of the Reagents 

Extraction 

0.1 M sodium pyrophosphate: dissolve 44.6 g of Na4P 2 O7,10H 2 O in 

distilled water and bring to 1 L; check the pH which must be 10.0. 

Clarification 

Superfloc cyanamid N-100 (cyanamid Corp. Gosport, Hampshire, UK): 
dissolve 0.2 g of superfloc in 100 mL of water and agitate in darkness for 
16 h with a PTFE magnetic bar stirrer, protect from the light in a brown 
bottle; a fresh solution should be made each week. 

Procedure 

With a laboratory precision balance, weigh one gram of soil (0.2 mm 
particle size) in a 250 mL polyethylene tube (with screw stopper). Add 
100 mL of 0.1 M sodium pyrophosphate reagent and agitate for 16 h at 
ambient temperature (20°C). 

Add 0.2 mL of superfloc and homogenize on a rotary shaker for 10 
min, then centrifuge at 20,000 g. With a millipore filter syringe remove 
from the quantities of supernatant needed to analyze the required 
elements (mostly Fe, Al, Si, and C). Add two blanks (with only reagents) 
and two reference samples in each series,. 

Measure the concentrations of Al, Fe, Si by plasma emission or atomic 
absorption spectrometry (cf. Chap. 31) with standards diluted in the 
extraction matrix. For measurements by absorption spectrocolorimetry, 
destroy the pyrophosphate matrix before measurement (cf. "Procedure" 
under "Acid Oxalate Method under Darkness"). 

Calculations 

Data collected: 

A, B, D, P,fas in other methods (cf. "Calculations" under Sect. 6.2.1). 



200 Mineralogical Analysis 

% contents (Fe, Al, Si) = 10 (A-B) DflP 

The results can be expressed in oxides (cf. "Calculations" under Sect. 
6.2.1). 

Remarks 

The method is well suited for differentiation of podzolic B horizons. The 
conditions governing centrifugation and clarification must be as 
homogeneous as possible, and the speed and time of centrifugation 
should be rigorously respected. If the series has to be stored before 
measurement by spectrography, store protected from the light in the 
refrigerator at 6-8 °C. 

The percentages of extracted organic carbon are often measured on 
automated CHN apparatuses (cf. Chap. 10) at the same time as Fe, Al and 
Si are measured using spectrographic techniques (cf. Chap. 31). Series of 
extractions whose action is due to a gradual increase in the pH of the 
medium are often used. These differential extractions make it possible to 
characterize increasingly resistant forms: 

- A preliminary extraction using 0.1 M sodium tetraborate buffered at pH 
9.7 6 enables the electrostatic linkages to be broken by simple exchange. 
The adsorbed organic molecules of low molecular weight are extracted. 
They contain complexed iron and aluminium that comprise the recently 
insolubilised "mobilizable complexes". The soil aggregates are not 
destroyed (Bruckert 1 970, 1 974, 1 979). 

-The 0.1 M pyrophosphate method at pH 10 then makes it possible to 
break the coordination linkages with the hydroxides and oxides in the 
coating on clays. 

- Lastly, the 0.1 M sodium hydroxide method at pH > 12 (cf. Sect. 6.2.5) 
enables the organo-mineral linkages to be destroyed, even those of the 
allophane-humic acid complexes. 



Preparation of reagent: 0.1 N sodium tetraborate pH = 9.7: dissolve 21 g of 
Na 2 B4O7,10H 2 O in approximately 900 ml_ of water; add 1.8 g of sodium 
hydroxide pellets; homogenize; check the pH which must be 9.7; bring to 1 L 
with deionised water. 

Extraction : 1 g of soil ground to 0.2 mm + 100 mL of 0.1 N sodium tetraborate 
solution; stir for 1 h; centrifuge at 20,000 g and continue as for pyrophosphate 
extracts (Al, Si, Fe, C, etc.). 



Selective Dissolution 201 

6.2.5 Extraction in strongly alkaline mediums 



Principle 

Methods using soda and sodium carbonate reagents are based on: 

- Dissolution in strongly alkaline medium of some silicon, aluminium, 
and aluminosilicate compounds; these can form soluble silicates and 
aluminates according to the simplified reaction: 

Al + NaOH + H 2 -> NaA10 2 + 3/2 H 2 . 

- concentration in the residue of insoluble compounds, and especially of 
iron and manganese. 

An attack using boiling 0.5 M sodium hydroxide solution for 2 min 30 s 
solubilizes organic forms of aluminium and silicon, hydrated non- 
crystalline and crystalline (gibbsite) aluminium oxides, opaline silica and 
diatoms, and finally amorphous or crypto-crystalline aluminosilicates like 
allophane and imogolite (Si0 2 :Al 2 03 ratio = 1.5-2.3). Some 1:1 silicates 
are attacked and partially dissolved. Iron compounds are not extracted. 

An attack using 0.5 M sodium carbonate for 16 h at 20°C (Follet et al. 
1965) has a more mitigated action and makes it possible to solubilize the 
organic and non-crystalline aluminium compounds as well as a certain 
proportion of gibbsite. Very finely divided siliceous compounds and 
opaline silica can be partially dissolved, amorphous aluminosilicates are 
solubilized, but allophane and imogolite are not completely solubilized. 
Phyllosilicates and iron compounds are not attacked. 

The iron compounds untouched by these two treatments can be studied 
in this enriched residue. But a more vigorous method with 5 M NaOH 
solutions and boiling for 2 h makes it possible to dissolve the majority of 
1:1 clays and clay minerals present and thus ensure a higher 
concentration. Despite this treatment, some minerals such as quartz, 
anatase, rutile, cristobalite, and some 2:1 clays may still be present in the 
residue. 

The action of the reagents mainly depends on the dispersion of the 
particles, the state of division of the silicon and aluminium substances, 
the crystallinity and reactivity of the surfaces of ordered or X-ray 
amorphous compounds. 

Extraction in a 0.5 M NaOH medium with a limited period of boiling 
enables differential solubilization of aluminium and silicon compounds as 
well as of organic matter with high reactive surface, whereas well- 
crystallized compounds are spared as their solubilization requires a much 
longer period of boiling. 

The solubility of aluminium hydroxides is amplified by an initial 
hydrolysis mechanism of the monomeric forms of aluminium (at low 



202 Mineralogical Analysis 

concentrations): A10H 2+ , Al(OH) 2 + , Al(OH) 3 , Al(OH) 4 ~, the latter existing 
only in an alkaline medium according to the reaction: 

Al 3+ + 4 H 2 -> Al(OH) 4 " + 4 H + 

Aluminium is in tetrahedral coordination. At higher concentrations, the 
polymeric forms gradually take the form of [Al 6 (OH)i 2 ,(H 2 0)i 2 ] 6+ units. 

In addition, all the acid groups of organic macromolecules (humic and 
fulvic acids) are dissociated, and the polar and anion sites are easily 
solvated. Under these operating conditions, most of the organomineral 
bonds are broken, even the very resistant ones between allophanic-Al and 
humic acid of Andosols. The poorly ordered aluminium and silicon 
compounds or their organic and inorganic derivatives pass in solution in 
aluminate or silicate form. 

Dissolution in sodium hydroxide solution can result in an oxidative 
medium by breaking down some humic acid forms in the presence of 
oxygen, which modifies the Fe 2+ :Fe 3+ ratios in the residues. Operating in 
nitrogen atmosphere can mitigate this phenomenon and also minimize the 
carbonation of sodium hydroxide by atmospheric C0 2 . 

Finally, in addition to the above processes, the strongly dispersing 
action of sodium hydroxide on the phyllosilicates also has to be 
considered. The elementary components of the stable micro-aggregates 
maintained in place by Fe, Al, Si oxide coating are first released by the 
dissolution process and then dispersed thereby increasing the action of 
the reagent at the solid-liquid interfaces. The alkaline character of the 
surfaces of the oxide decreases according to the series: 





Amorphous 


>y- 


> a- 


>Y- 


>y- 




hydrated Al 


AIO(OH) 


Al(OH) 3 


Al(OH) 3 


A1A 




oxides 


boehmite 


bayerite 


gibbsite 




pHat 


9.45 


9.40 


9.20 




8.00 


isoelectric 












point 













In addition to sodium hydroxide and carbonate, and sodium or 
potassium pyrophosphate, other reagents that have been used to extract 
the organic matter and organomineral complexes are: ethylene diamine 
(EDA), NN-dimethylformamide, sulfolane, pyridine, dimethyl sulfoxide, 
etc. 



Selective Dissolution 



203 



Reagents 

The three mostly widely used reagents are described here as they 
correlate well with other selective extraction methods and characterize 
the resistance to the dissolution of the aluminous or siliceous products 
satisfactorily. Many alternatives have been proposed whose relative 
effectiveness (but not the degree of selectivity) is roughly classified in 
Table 3. Potassium hydroxide solutions are sometimes used instead of 
sodium hydroxide. 

Table 6.3. Strongly alkaline reagents classified in order of decreasing efficiency 



5 mol 



> 0.5 mol 



(NaOH)L" 1 (NaOH)L" 1 
pH>14 pH>12 



s. a or i > 0.5 mol 
> 1.25 mol 

n\laOl-h|- 1 ( Na 2C0 3 ) 

(Na0H)L pH10.7 



> 0.5 mol 
(Na 2 C0 3 )L~ 
pH 10.7 



>0.1 mol 
(NaOH) L" 



boiling 



boiling 



80°C 



boiling 



20°C 



20°C 



2h 



2.5 min 



20 min (or 
shorter) 



1 h(or 

variable 

times) 



16 h 



iron 
enrichment 



selective 
extraction of 
free oxides 
allophane 
imogolite 



eliminates 
eliminates inter-particle 
gibbsite and cements, 
free oxides solubilizes 1:1 

minerals 



selective 

extraction of clay 
allophane dispersion 
imogolite 



Norrish and Hashimoto 
Taylor and Jackson 

(1961) (1960) 



Folletetal. 
(1965) 



Preparation 



5 mol (NaOH) L" 1 solution: carefully dissolve 200 g of analytical grade 
sodium hydroxide pellets in 1 L of distilled water previously boiled to 
eliminate carbon dioxide Leave to cool with no contact with air and 
store in a polythene bottle. Prepare fresh solution every week. 



204 Mineralogical Analysis 

- 0.5 mol (NaOH) L 1 solution: dissolve 20 g of sodium hydroxide pellets 
in 1 L of previously boiled distilled water. Store in a polythene bottle. 
This reagent should be freshly prepared every day. 

- 0.5 mol (Na 2 C0 3 ) L" 1 solution: dissolve 53 g of anhydrous Na 2 C0 3 in 1 
L of distilled water and store in a polythene bottle. 

- 0.5 mol (HC1) L _1 : take 42 mL of HC1 </= 1.19 and bring to 1 L. 

Procedures 

Selective dissolution with 0.5 M Na 2 C0 3 at 20°C 

(Folletetal. 1965): 

- Weigh 100 mg of soil sample ground to 0.2 mm and place in a 100 mL 
centrifuge tube. 

- Add 80 mL of 0.5 M Na 2 C0 3 solution; close the tube and shake for 16 h 
with a rotary shaker. 

- Centrifuge at 5,000 g for 10 min. 

- Transfer the supernatant in a 200 mL volumetric flask. 

- Wash the centrifugation pellet with distilled water and recentrifuge. 

- Add to the previous extract and bring to 200 mL with deionised water. 

- Carry out spectrometric measurements on the extract without delay 
using atomic absorption or inductively coupled plasma emission (Si and 
Al). 

Selective dissolution with boiling 0.5 M NaOH 
(Hashimoto and Jackson 1960): 

- Weigh 100 mg of soil sample ground to 0.2 mm (or use the residue of a 
DCB extraction) in a 250 mL nickel or PTFE crucible. 

- Add 100 mL of 0.5 M NaOH boiling solution and homogenize. 

- Maintain boiling for 2 min 30 s. 

- Rapidly cool and transfer in a 250 mL polythene centrifuge tube and 
centrifuge for 10 min at 5,000 g 

- Transfer the supernatant in a 500 mL volumetric flask. 

-Wash the centrifugation pellet with distilled water, recentrifuge, and 
add the rinsing solution in the volumetric flask. 

- Adjust to volume with distilled water and measure Si and Al without 
delay using atomic absorption spectrometry or plasma emission 
spectrometry. 



Selective Dissolution 205 

Dissolution with boiling 5 M NaOH (2 h) 
(Norris and Taylor 1961): 

- Weigh 100 mg of sample ground to 0.2 mm (or use the residue of other 
selective extractions) in a stainless steel or PTFE beaker. 

- Add 100 mL of 5 mol (NaOH) L" 1 solution. 

- Boil for 2 h. 

- Cool and centrifuge at 5,000 g for 10 min. 

- Decant the supernatant (which can be discarded or analysed). 
-Wash the centrifugation pellets with a little water, then wash three 

times with 0.5 mol (HO) L" 1 solution to dissolve the resulting sodalite 
and to eliminate sodium chloride, then wash again with water until 
negative reaction of chlorides. 

Dry the sample ready for the analysis of manganese and iron oxides, 
check the absence of kaolinite and sodalite by XRD. 

Calculations 

All the results are expressed in oxides (cf. "Calculations" under Sect. 
6.2.1). 

Remarks 

The NaOH:Al ratio influences the development of aluminium hydroxides 
(Hsu 1977). It is thus important to standardize the procedures during the 
Al precipitation. Studies of 27 A1 by nuclear magnetic resonance show 
that, for a given concentration and pH, the nature of the gel gradually 
changes with ageing (Hsu 1984). These modifications, which occur in the 
natural environment, also occur in the synthetic mediums though at a 
different scale (Stol et al. 1976). NMR spectrometry is a particularly 
powerful tool to differentiate suspended or dissolved polynuclear species. 
Sometimes the modifications observed can explain such apparently 
induced variations by separation techniques such as centrifugation, 
ultrafiltration, or dialysis. Consequently it is better to carry out 
spectrometric measurements on products that have been recently 
extracted by different methods. 

For the 5 mol (NaOH) L" 1 reagent, Kampf and Schwertmann (1982) 
recommend the boiling extraction method and in the presence of gibbsite 
to modify the reagent which must contain 0.2 mol (silica) L" 1 in order to 
minimize phase changes and a possible increase in crystallinity, and to 
inhibit dissolution and recrystallization of substituted aluminium in 
goethites. Ferrihydrite, which can be converted into hematite and/or 
goethite, remains almost intact. However, the increase in the silica 
content results in more abundant precipitation of sodium and aluminium 



206 Mineralogical Analysis 

silicate (sodalite) which must be eliminated from the residue by several 
washings with 0.5 mol (HO) L _1 solution. 

The method of concentration with 5 mol (NaOH) L" 1 makes it possible 
to identify manganese oxides in the residue more clearly. These oxides 
are generally found at low concentrations and display weak crystallinity 
in soils. Birnessite and lithiophorite are found with iron oxides. Birnessite 
can be dissolved in hydroxylamine chloride as goethite and lithiophorite 
are not affected by this treatment. A final attack using the DCB method 
makes it possible to dissolve the goethite (Shuman 1982; Tokashiki et al. 
1986). 

In the method based on boiling soda for 2 min 30 (cf. "Selective 
dissolution with boiling 0.5 M NaOH"), time is critical and must be 
carefully respected to avoid excessive solubilization of kaolinite and 
halloysite which can gradually pass in solution. The chlorites and 
montmorillonites, which were previously heated to 500°C, are not much 
affected. Spectrometric measurements should be made rapidly after 
extraction to avoid ageing of the extracts and precipitation of 
aluminosilicate. 

A large volume of solution compared to the weight of soil sample 
should be used to avoid the saturation of the extracts by aluminium and 
silicium. To avoid contamination, only PTFE, stainless steel, or nickel 
laboratory equipment is recommended. Pyrex glass can cause pollution 
by Si, Al, Fe. 

Dissolution of the gibbsite can be corrected if it is measured by DTA 
(cf. Chap. 7). 

Dissolution with 0.5 mol (NaOH) L" 1 solution at boiling point does not 
enable very fine differentiation between amorphous and crystalline 
products because of the sensitivity of poorly ordered 1:1 clays and 
gibbsite. However, correlation with the oxalate method is generally good. 



6.3 Other Methods, Improvements and Choices 



6.3.1 Differential Sequential Methods 



Principle 

Many methods have been developed. When used in sequence, the 
methods described in Sect. 6.2 earlier can be considered as sequential 
multi-reagent methods. 



Selective Dissolution 



207 



The Segalen (1968) method uses an alternating process of hydrolysis 
and protonation in cold acid medium (without complexation or reduction) 
to solubilize some iron and aluminium compounds, then a treatment in 
hot alkaline medium (80°C) to solubilize the aluminium and silica 
compounds (cf. Sect. 6.2.5 earlier). These treatments are alternated 
several times to establish cumulative curves of quantity vs time (Fig. 6.7). 

Fractionation expresses the differences in the solubility of compounds 
in these mediums 7 . Solubilization kinetics and hydrolysis constants 
depend on (1) the type, nature and concentration of the reagents, the 
soil/solution ratio and temperature and (2) nature and size of the sample, 
its elementary particle content, the degree of crystallinity and the extent 
of specific surface of clay minerals, the molecular arrangements and the 
degree of substitutions. 



"O 

c 

13 

o 

Q. 

E 
o 
o 

■D 

B 

O 

X 

<D 



Amorphous + crystallized | 




Hours 







Amorphous 



D E F G H 
Extraction sequences 



Fig. 6.7. Cumulative curve of extraction 

The cumulative curve of the extracted compounds integrates these 
parameters (1) the lower part of the curve shows a parabolic segment that 
rises to a greater or lesser extent depending on the soil types which 
correspond mainly to the rapid dissolution of non-crystalline products 



For example the dissolution kinetics of the iron forms in 0.5 mol (HCI) L" 
solution at 25°C (Shidhu et al. 1981) follows the series: (ferrihydrite, feroxyhite > 
lepidocrocite > magnetite > akaganeite > maghemite > hematite > goethite). 



208 Mineralogical Analysis 

with a very high specific surface area (Fig. 6.7); the kinetics of 
dissolution shows that a fraction of very fine crystalline products can also 
pass in solution in the same time interval; (2) the rectilinear upper 
segment has a weak slope indicating the weak dissolution of the well- 
crystallized and chemically not very reactive products. 

A gradual change in the slope may indicate the end of dissolution of the 
very fine crystalline particles, or on the contrary, the appearance of 
practically non-ordered zones of preferential dissolution. Extrapolation of 
the upper segment at the intersection with the ^-coordinate provides an 
estimate of the percent of amorphous substances. 

Reagents 

The reagents recommended by Segalen (1968) for the acid attack are 2, 4, 
and 8 mol (HO) L" 1 hydrochloric acid solutions (the last concentration 
was finally retained) and for the alkaline treatment, boiling for 5 min 
with 0.5 mol (NaOH) L" 1 solution. The extraction sequence comprises 8 
alternating acid-alkaline treatments: 

- 8 mol (HC1) L" 1 : in a 1 L volumetric flask, add 830 mL of reagent grade 
hydrochloric acid to about 100 mL distilled water, agitate, leave to cool 
and bring to 1 L; alternatively if 6, 4, 2, or 0.5 mol (HO) L" 1 solutions 
are used, add respectively, 623, 415, 208, or 52 mL hydrochloric acid 
in the 1 L flask. 

-0.5 mol (NaOH) L" 1 : add 20 g of NaOH pellets in a 1 L volumetric 

flask, dissolve in distilled water and bring to 1 L. 

The proposed modifications of this method concern the temperature 
(from ambient to boiling), the duration of contact and finally the use of 
only one reagent (2 mol (HO) L" 1 ) without alternating with NaOH. 

Procedure 

(Segalen 1968): 

-Weigh 500 mg of soil (ground to 0.2 mm) in a 100 mL polyethylene 
centrifuge tube. 

- Add 50 mL of 8 mol (HO) L" 1 solution, homogenize and leave in 
contact for 30 min at room temperature. 

- Centrifuge at 5,000 g for 10 min. 

- Transfer the supernatant in a 100 mL volumetric flask. 

- Add 45 mL of deionised water, resuspend the centrifugation pellet by 
Vortex agitation. 

- Centrifuge at 5,000 g and add the washing water in the volumetric 
flask. 



Selective Dissolution 209 

-Complete to 100 mL; this is extract A (acid) for the measurement of 
iron, aluminium, and silicon. 

- Add 50 mL of NaOH 0.5 mol L" 1 solution in the centrifuge tube and 
resuspend. 

- Place in the boiling water bath for 5 min. 

- Centrifuge at 5,000 g for 10 min. 

- Collect the supernatant in a 100 mL volumetric flask. 

- Wash the residue with 45 mL distilled water and add the washing water 
to the extract; complete to 100 mL; this is extract B (alkaline) for the 
measurement of alumina and silica. 

Repeat the double extraction process eight times. 

Calculations 

All the results obtained by atomic absorption or inductively-coupled 
plama emission spectrometry are expressed in per cent of oxides (cf. 
"Calculation" in Sect. 6.2.1). 

For alumina and silica, the results obtained on extracts A and B must 
be added. For iron, the acid solution is used alone. Cumulative curves of 
iron, alumina, and silica are established (Fig. 6.7); the tangent to the right 
of the curve gives the percentage of amorphous compounds. 

Remarks 

The procedure using the above concentrations (alternating 8 mol (HC1) L" 1 
and 0.5 mol (NaOH) L" 1 with 5 min boiling), was found to be too 
energetic and insufficiently selective for many pedological situations and 
many minerals as the amorphous substances were often over-estimated. 
This method thus needs to be adapted to the type of soil being analysed. 
The original unmodified method dissolves amorphous iron and 
complexed iron, but if the use of very aggressive reagents results in the 
rapid dissolution of the amorphous substances, it also solubilizes 
crystallized products: 1:1 clays, mica, chlorite, biotite, hornblende, 
nontronite, gibbsite, etc. (Colmet-Daage et al. 1973; Quantin and 
Lamouroux 1974; Quantin et al. 1975; Yong et al. 1979; Bentley et al. 
1978, 1980; Quigley et al. 1980, 1985; Torrance et al. 1986). 

The degree of selectivity may be insufficient for certain soils and 
amorphous substances will be considerably over-estimated. On their own, 
the kinetics curves do not show which forms are truly solubilized. 

The kinetics of dissolution is significantly correlated with the 
concentration of the reagents, and to time and temperature. Modifying 
these parameters can significantly change the dissolution kinetics and 
selectivity. The results always have to be linked with a precise procedure. 



210 Mineralogical Analysis 

As is true for all the other methods, an excess of reagent is necessary to 
avoid the effects of saturation. This method is not really suitable for 
repetitive analysis without robotization because of the length of the 
successive extractions. Six double extractions can be carried out each day 
on a small series. 

The study of goethite dissolution (Cornell et al. 1974, 1975, 1976; 
Schwertmann et al. 1985) in 0.5 mol (HC1) L" 1 at 20 and 60°C using 
transmission electron microscopy (TEM) shows that preferential zones of 
dissolution create new surfaces that can give sigmoid curves of 
dissolution vs time. This mechanism of dissolution is related to the 
structure of the molecular configurations. The kinetics of dissolution of 
goethite is slowed down by a strong Al 3+ substitution. These studies 
highlight the changes in the concepts of dissolution and of "amorphous" 
substances as well as the need to check the size and the state of surfaces 
of minerals using SEM and XPS microscopy. 

6.3.2 Selective Methods for Amorphous Products 

Many other methods are used for the description of amorphous 
substances, in particular the release of hydroxyls by fluorides and 
trimethylsilylation. 

Release of Hydroxyls by Fluorides 

This method is based on the general reaction: 

(mineral-OH) + F" ^ (mineral-F) + OH 

which satisfactorily accounts for the reactivity of the crypto-crystalline 
components, aluminosilicates like allophane or imogolite, or amorphous 
aluminium forms, the reactions being very rapid. 

Al (OH) 3 + 6NaF -+ Na 3 AlF 6 + 3NaOH 

The above reaction made it possible to develop a NaF field test for 
andosols. The total released OH" correlates well with the amorphous 
forms of Al and Si, with a weak influence of Fe. Quantification is 
performed by titration at constant pH: 
- Put 25 mg of soil ground to 0.2 mm in contact with 0.85 mol (NaF) L" 1 

solution solution at pH 6.8 and at 25°C; 



Selective Dissolution 211 

-Using a titrimeter, maintain the pH at 6.8 for 30 min by adding a 

titrated acid solution. The quantity of acid required for neutralization 

provides an estimate of the non-crystalline substances. 

The reaction can be performed stoechiometrically only in the absence 

of organic matter and carbonates. The crystalline substances are not 

sufficiently reactive to react significantly in such a short period of time. 

Thmethylsilylation 

This method is specifically used for silica compounds; it is derived from 
organic chemistry and is based on the reaction of the silicic acid with 
organo-silicic compounds which produce stable and volatile organosilyl 
derivatives that can be separated and identified by gas chromatography. 
The basic reaction can be written briefly: 



M 2 H Si(CH 3 ) 3 

° HCI 9 (CH 3 ) 3 SiOH ,^ uxrv 9 

M 2 -Si - OM 2 — > H O -Si - O H - — ► (CH 3 ) 3 Si O -Si - O Si(CH 3 ) 3 

6 6 6 

M 2 H Si(CH 3 ) 3 

Monomelic phase Silicic Trimethylsilyl derivatives 

with o-silicon bonds acid 



This reaction makes it possible to determine the degree of 
polymerization of silica and to link it to different types of deterioration. 
Inorganic gels that are rich in aluminium are particularly reactive. 
Crystalline substances are not very active. 

Place 50 mg of soil sample in contact with 9 mL of hexamethyl 
disiloxane (HMDS) 8 at 4°C in 0.2 mL of water and an internal standard 
(n-Eicosane, CH 3 (CH 2 )i8CH 3 ). After homogenisation, add 2 mL 
trimethylchlorosilane (TMCS) 8 at 4°C. The hydrolysis releases 
hydrochloric acid which reacts with the soil amorphous silicates by 
exchange of cations and protonation. Leave in contact for 16 h at 4°C. 
The silicic acid forms trimethylsilyl derivatives. 



3 TMCS, Trimethylchlorosilane (CH 3 ) 3 SiCI; HMDS, Hexamethyldisiloxane 
(CH 3 )3Si-0-Si(CH 3 )3. 



212 






Mineralogical Analysis 


Table 6.4. 


Approximate balance of soil mineral phases 


from different extractions 


Fe forms 


symbol 


description 


Al, Si, Mn, and 
others 



total iron 



Fe(T) 



- analysis essential for the 
establishment of geochemical 
and mineralogical balances and 
characteristic ratios 

- the method of attack by 
boiling HCI is insufficient for 
satisfactory quantification. An 
alkaline fusion should be 
carried out. Minerals such as 
pyrites, marcasite, and 
magnetite are difficult to 
mineralize. 

ratios 

Fe(T)/Clays 

Fe(CBD)/Fe(T) are indices of 

weathering of the horizons. 

Degree of evolution of 

hydromorphic soils. 



free iron 



Fe(f) = 
Fe(DCB) 



crystaline 
free iron 



iron extractable by DCB 
reagent (cf. Sect. 6.2.2): iron 
mobilizable in pedogenetic 
processes. 

This iron is of pedological 
interest since it is not linked 
with the structure of the silicate 
lattices (clays, primary 
minerals) that presents different 
hydroxylated and hydrated 
forms with a varying degree of 
crystal I in ity. 
ratios 

Fe(f)/Clays, Fe(oxda)/Fe(DCB) 
indicate the degree of 
weathering and crystallinity of 
free oxides and concretions. 
Fe(DCB)/Fe(T) is the index of 
deterioration and mobility of 
iron in a profile. 
Fe(DCB)-Fe(oxda) 



total free Al 
(exchange acidity, 
exchangeable Al + , 
ECEC, etc.), marker 
of deterioration 
(acidolyse, 
complexolyse) 

free Mn 



iron in Fe(sil) Fe(T)-Fe(DCB) 
silicates the iron in silicates originating 
from primary minerals and the 
lattices of phyllitic minerals; 
a reduction indicates 
progression in deterioration 
(cambic horizon) 



Selective Dissolution 



213 



not well- 
crystallized 
iron oxides 



Fe(OA) Fe(oxda)-Fe(EDTA) 



well- 
crystallized 
iron oxides 



Fe(OC) 



Fe(DCB)-Fe(oxda) 
the well-crystallized iron oxides 
are identifiable using 
instrumental techniques (XRD, 
DTA, etc.). These oxides are 
inherited or newly formed. The 
presence of lepidocrocite and 
magnetite which are sensitive 
to the oxalate treatment may 
affect the results. Magnetite is 
inherited and is not formed 
during pedogenesis. 



iron in 
amorphous 
forms and 
organic 
complexes 



Fe(pyro) immovable complex 



AKtetraVAKPvro) 
decreases with 
ageing of the gel 
(soluble aluminates 
in alkaline medium) 



complexed 
iron 



Fe(tetra) 



mobile or mobilizable complex. 
Method used in sequence with 
reagents of increasing 
efficiency: 
Tetraborate pH 
9.7<pyrophosphate pH 9.8 < 
NaOH pH 12 
- Fe(tetra):Fe(pyro) 
characterizes the degree of 
evolution of complex Fe- 
organic matter, if Fe(tetra) 
decreases and Fe(pyro) 
increases there is pedogenesis 
with strong biological 
deterioration. 



Fe(oxda) iron extracted by oxalate in 
darkness (cf. Sect. 6.2.1 
above), 

if Fe(oxob), Fe(EDTA), 
Fe(pyro) are weak there is 
weak deterioration 



Add 2 mL H 2 to hydrolize the excess of TMCS. Centrifuge to 
separate solid and liquid phases and transfer the organic solution on 1 g 
cation exchange resin (Amberlite 15). After 5 h of contact and filtration, 



214 Mineralogical Analysis 

separate the extract and quantify the trimethylsilyl derivatives by gas 
chromatography with nitrogen as carrier gas. 

6.3.3 Brief overview of the use of the differential methods 

There are many publications concerning each method of extraction 
making it possible to find examples of the use of all the methods, either 
alone or in comparison with other methods, e.g. Aomine and Jackson 
(1959), Bruckert (1979), Jeanroy (1983), Baize (1988), Krasnodebska- 
Ostregaetal. (2001). 

The final objective may simply be the elimination of substances (e.g. 
cementing agents) that obstruct dispersion or instrumental observation 
(e.g. XRD or IR spectra). In this case, it is not essential to identify the 
soluble products; on the contrary, to understand the mechanisms of 
mineralogical and pedogenic evolution both the soluble phases and the 
solid phases have to be taken into account. Comparisons should be made 
of results expressed in per cent of oxides; this is a practical way to 
homogeneously quantify the mineralogical phases but does not 
correspond to the chemical forms actually present in the soil. 

The combination of several methods makes it possible to establish an 
approximate balance of the different aluminium and iron forms in the soil 
(Table 6.4). Isolated entities are often not sufficiently specific and results 
are somewhat empirical, but give a precise enough view of the 
weathering phenomena. In general, variations in extracted contents of a 
given element (initially iron, then aluminium, silica, and manganese) are 
compared on the same sample first by total analysis and then by selective 
analyses (free forms, amorphous forms, etc.). Next, variations between 
horizons of a soil profile and finally variations between profiles can be 
compared. Correlations with size and behaviour of components can be 
highlighted. Iron is the main element used as a tracer of deterioration. It is 
insoluble in the ferric oxidation state and a loss generally indicates a 
pedogenic process of reduction, also indicated by the colour of the soil. 
The processes of biochemical humification result in the formation of 
iron-organic complexes. 

In andosols, Al and Si, components of allophane and imogolite 
(Fig. 6.1), are measured first. In these soils, allophanic materials can 
sometimes be estimated indirectly, for example by the measurement of 
the cation exchange capacity on a sample from which the organic matter 
has previously been eliminated; one part of the sample is treated with a 



Selective Dissolution 215 

sodium carbonate solution for 60 min, and another part with a boiling 
sodium acetate solution for 15 min, and the result will be a difference in 
cation exchange capacity that correlates well with allophane content 
(Aomine and Jackson 1959). 

An instrumental technique, differential diffractometry, makes it possible 
to evaluate the action of the dissolution reagents and the behaviour of soil 
materials. Several diffraction spectra of a sample are made before and 
after treatment by selective dissolution. An appropriate computer 
program can be used to calculate the difference between the spectrum of 
the treated sample and the spectrum of the untreated sample. Quartz or a- 
alumina is used as an internal standard to calculate the scale factor for the 
subtraction of the spectra. This enables the spectrum of the dissolved 
substances to be reconstructed during selective treatment (Campbell and 
Schwertmann 1985). 



References 

Alexsandrova LN (1960) The use or pyrophosphate for isolating free humic 

substances and their organic -mineral compounds from the soil. Soviet 

Soil Sci., 190-197 
Aomine S and Jackson ML (1959) Allophane determination in ando soils by 

cation exchange delta value. Soil Sci. Soc. Am. Proc, 23, 210-214 
Atkinson RR, Posner AM and Quirk J (1968) Crystal nucleation in Fe (III) 

solutions and hydroxyde gels. J. Inorg. Nucl. Chem., 30, 2371-2381 
Avery BW and Bascomb CL (1982) Soil Survey Laboratory Methods., Soil 

Survey of England- Wales (Harpenden), 6 
Baize D (1988) Guide Des Analyses Cour antes En Pedologie: Choix - 

Expression - Presentation - Interpretation., INRA, 172 pages 
Ballantyne AKD, Anderson DW and Stonehouse HB (1980) Problems 

associated with extracting Fe and Al from Saskatchewan soils by 

pyrophosphate and low speed centrifugation. Can. J. Soil Sci., 60, 141— 

143 
Bentley SP, Clark NJ and Smalley IJ (1980) Mineralogy of a Norwegian 

postglacial clay and some geotechnical implications. Can. Miner., 18, 

535-547 
Borggaard OK (1976) Selective extraction of amorphous iron oxide by EDTA 

from a mixture of amorphous iron oxide, goethite and hematite. J. Soil 

Sci., 27, 478-486 



216 Mineralogical Analysis 

Bruckert S (1979) Analyse des complexes organo-mineraux des sols. In 

Pedologie 2, constituants et proprietes du sol, Bonneau M. and 

Souchier B. ed. Mason, IX, 187-209 
Cambier P and Sposito G (1991) Adsorption of citric acid by synthetic 

pseudoboehmite. Clays Clay Miner., 39, 369-374 
Campbell AS and Schwertmann U (1985) Evaluation of selective dissolution 

extractants in soil chemistry and mineralogy by diferential X-Ray 

diffraction. Clay Miner., 20, 515-519 
Colmet-Daage F, Gautheyrou J, Gautheyrou M and De Kimpe C (1973) Etude 

des sols a allophane derives de materiaux volcaniques des Antilles et 

d'Amerique latine a l'aide de techniques de dissolution differentielle. 

Iere partie. Etude des produits solubilises. Cah. ORSTOM serie Pedol, 

XI, 97-120 
Cornell RA, Posner AM and Quirck J.P (1976) Kinetics and mechanisms of the 

acid dissolution of goethite (oc-Fe OOH). J. Inorg. Nucl. Chem., 38, 

563-567 
Cornell RM and Schindler PW (1987) Photochemical dissolution of goethite in 

acid/oxalate solution. Clays and clay Miner., 35, 347-352 
Cornell RM, Posner AM and Quirck J.P (1974) Crystal morphology and the 

dissolution of goethite. J. Inorg. Nucl. Chem., 36, 1937-1946 
Cornell R.M., Posner A.M and Quirk JP (1975) The complete dissolution of 

goethite. J. Appl. Chem. Biotechnol., 25, 701-706 
De Endredy AS (1963) Estimation of free iron oxides in soils and clays by a 

photolytic method. Clay Miner. Bull, 29, 209-217 
Deb BC (1950) The estimation of free iron oxides in soils and clays and their 

removal. J. Soil ScL, 1, 212-220 
Duchaufour Ph and Souchier B (1966) Note sur une methode d'extraction 

combinee de 1' Aluminium et du fer libres dans les sols. ScL du Sol, 1, 

17-29 
Farmer VC and Fraser AR (1978) Synthetic imogolite, a tubular hydroxy- 

aluminium silicate. In International Clay Conference., Elsevier, 

Amsterdam, 547-553 
Farmer VC, Fraser AR and Tait JM (1979) Characterization of the chemical 

structures of natural and synthetic aluminosilicate gels and soils by 

infrared spectroscopy. Geochim. Cosmochim. Acta, 43, 1417-1420 
Follett EAC, McHardy WJ, Mitchell BD and Smith BFL (1965) Chemical 

dissolution techniques in the study of soil clays. Clay Miner., 6, 23-43 
Franzmeier DP, Hajek BF and Simonson C.H (1965) Use of amorphous material 

to identifiy spodic horizons. Soil Sci. Soc. Am.Proc, 29, 737-743 
Hashimoto I and Jackson ML (1960) Rapid dissolution of allophane and 

kaolinite and halloysite after dehydratation. Clays clay Miner., 1, 102- 

113 
Henry S (1958) Synthese de quelques oxydes de fer au laboratoire. CR. duXXXI 

Congres intern, de Chimie Indus trielle (Liege)., Mercurius, 1-3 
Hetier JM and Jeanroy E (1973) Solubilisation differentielle du fer, de la silice 

et de l'alumine par le reactif oxalate-dithionite et la soude diluee. 

Pedologie, 23, 85-99 



Selective Dissolution 217 

Holmgren GGS (1967) A rapid citrate-dithionite extractable procedure. Soil Sci. 

Soc. Am. Proc, 31, 210-211 
Hsu PH (1977) Aluminium hydroxydes and oxyhydroxyde. In Minerals in Soil 

Environments, Dixon JB Weed SB and ed., Soil Sci. Sc. Am., 99-143 
Hsu PH (1984) Aluminium hydroxides and oxyhydroxides in soils: recent 

developents. Annu. Meeting and Am. Soc. Agron 
Jeanroy E and Guillet B (1981) The occurence of suspended ferruginous 

particles in pyrophosphate extracts of some soil horizons. Geoderma, 

26,95-105 
Jeanroy E (1983) Diagnostic des formes dufer dans les pedogeneses temper ees. 

Evaluation par les reactifs chimiques d' extraction et apports de la 

spectrometrie Mossbauer. (etudes des formes organiques du fer 

amorphe dans les sols)., These Doctorat, Nancy, 109-129 
Kampf N and Schwertmann U (1982) The 5M-NaOH concentration treatment 

for iron oxides in soils. Clays clay Miner., 30, 401-408 
Klamt E (1985) Reports of meetings. Iron in soil and clay minerals. Bad 

Windesheim, West germany, July 1-13 1985. Bull. Soc. Int. Sci. du Sol, 

2,9 
Krasnodebska-Ostrega B, Emons H and Golimowski J (2001) Selective leaching 

of elements associated with Mn-Fe oxides in forest soil, and 

comparison of two sequential extraction methods. Fresenius J. Anal. 

Chem., 371, 385-390 
Kwong KF and Huang PM (1979) The relative influence of low-molecular- 
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Aluminium. Soil Sci., 128, 337-342 
Lewis DG and Schwertmann U (1979) The influence of Al on iron oxides. Part 

III - Preparation of Al goethites in M KOH. Clay Miner., 14, 1 15-126 
Lewis DG and Schwertmann U (1979) The influence of Al on the formation of 

iron oxides. Part IV: The influence of [Al], [OH] and temperature. 

Clays clay Miner., 27, 195-200 
Loveland PJ and Bullock P (1976) Chemical and mineralogical properties of 

brown podzolic soils in comparison with soils of other groups. J. Soil 

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Loveland PJ and Digby P (1984) The extraction of Fe and Al by 0,1 M 

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35, 243-250 
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13-22 
Mc Keague JA (1967) An evaluation of 0,1 M pyrophosphate and 

pyrophosphate-dithionite in comparison with oxalate as extractants of 

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Sci., 47, 95-99 



218 Mineralogical Analysis 

Neaman A, Mouele F, Trolard F, Bourrie G (2004a) Improved methods for 

selective dissolution of Mn oxides : applications for studying trace 

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Neaman A, Waller B, Mouele F, Trolard F, Bourrie G (2004b) Improved 

methods for selective dissolution of manganese oxides from soils and 

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509-518 
Schuppli PA, Ross GJ and McKeague JA (1983) The effective removal of 

suspended materials from pyrophosphate extracts of soil from tropical 

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dans certains sols a hydroxydes tropicaux. Cahiers ORSTOM Serie 

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Selective Dissolution 219 

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511-520 



Thermal Analysis 



7.1 Introduction 



7.1.1 Definition 

Many different analyses of phase transformations involve the use of 
temperature. This is true in the case of simple gravimetric measurements 
after drying at a given temperature (cf Chap. 1), or after chemical 
precipitation and drying to constant weight. The generic term "thermal 
analysis" generally only applies to methods carried out according to a 
dynamically controlled thermal programme making it possible to reveal 
and quantify different physicochemical transitions. The most common 
methods used in soil analysis record transformations by means of the 
temperature either of mass, energy, or the mechanical properties of the 
samples (Fig. 7.1). 

Measurements of Mass Variations 

The abbreviations TGA or TG stand for thermogravimetric analysis and 
DTG stands for differential thermogravimetry. Losses occur in the form 
of gases that are simple to detect by (evolved gas detection EGD) and to 
analyze by (evolved gas analysis EGA). 

Measurements of Variations in Energy 

These measurements mainly use DTA, differential thermal analysis and 
DSC, differential scanning calorimetry. They enable quantification of 
exothermic or endothermic energy changes at each temperature without 
inevitably modifying weight. 



222 



Mineralogical Analysis 



Measurements of Dimensional or Physical Variations 

These include TMA, thermo mechanical analysis (dilatometry) and 
DMA, dynamic mechanical analysis (viscoelasticity). Though these 
techniques are used especially in the field of ceramics and plastics, they 
are also suitable for the study of physical soil properties and specifically 
for the shrinkage and swelling linked to aggregation and water storage 
properties (Braudeau 1987, 1988; Braudeau et al. 1999, 2004). 



Transitions in thermal analysis 



Mass 
changes 



TGA- DTG 



Multi- 
component 
STA 



Energy 
changes 



— I 

Dimensional 
or mechanical 
changes 



A- DSC 



TMA- DMA 



Evolved 
gas analysis 
EGA 



1 

Optical 

magnetic 

electric 

sound, 

etc. 

changes 

in properties 



Fig. 7.1. Table summarizing the principal methods of thermal analysis: TGA, 
thermogravimetric analysis; DTG, differential thermogravimetry; DTA, 
differential thermal analysis; DSC, differential scanning calorimetry; 
TMA, thermomechanical analysis; DMA, dynamic mechanical 
analysis; STA, simultaneous thermal analysis; EGA, evolved gas 
analysis. The International Confederation for Thermal Analysis (ICTA) 
uses the term "derivation" for data resulting from mathematical 
transformations (derivative calculations on initially measured data) and 
"differential" for experimental measurements of changes in the delay 
step concerned 



Variations in properties (optical, magnetic, electric, or sound) also 
occur and can be measured analytically (for example, thermo- 
luminescence is widely used for dating), the loss of magnetic property is 
represented by the point of Curie, etc. 

The equipment used for these analyses has reached a high degree of 
sophistication thanks to its industrial use in the fields of ceramics, glass, 
cement, plaster, explosives, radioisotopes, pharmaceutics, and polymers 
where it is widely used for research and quality control. The simplest 



Thermal Analysis 223 

equipment is not very expensive, but top-of-the-range apparatuses 
(multiparametric, high temperature, high pressure, etc.) with their 
associated peripherals and data processing software, can be very costly. 



7.1.2 Interest 

Used in combination with XRD (cf. Chap. 4), IR (cf. Chap. 5) and other 
chemical analyses, thermal analysis techniques are invaluable tools in 
mineralogy, geology, pedology, soil chemistry, and physics. They allow 
the qualitative identification and quantitative analysis of clays (Table 7.1) 
and many minerals, and also the identification of the different forms of 
water in soils, oxidation of all forms of organic matter and inorganic 
materials, phase transitions, etc. 

The sensitivity of the DTA-DSC methods makes it possible to detect 
the presence of minerals at the limit of detection of XRD (e.g. goethite, 
gibbsite at concentration less than 0.25%, substances with short distance 
atomic arrangement) in clays and soils. 

All the techniques used enable the balance of the mineral 
transformations to be established as a function of different geochemical 
processes both in a weathering profile and in a topographic sequence. 

Reasonably precise quantitative analysis is possible of hydroxides and 
oxyhydroxides of iron and aluminium, as well as of clays and particularly 
of 1:1 kaolinite and halloysite. Continental sediments containing organic 
complexes can be studied by controlled oxidizing or non-oxidizing 
pyrolysis, and the evolved gases are analyzed by Fourier transform infra- 
red spectrometry (FTIR). DSC can quantify enthalpy changes during 
dehydration, dehydroxylation and other forms of structural 
decomposition in a thermal field extending from -150 to +725°C. The 
analysis of evolved gases by FTIR or mass spectrometry during 
temperature scanning makes it possible to calculate the exact chemical 
transformations undergone by the sample. 

Thermal analysis is particularly useful for the study of soil genesis, the 
study of soils rich in /?ara-crystalline compounds (e.g. andosols, 
histosols) and characterization of the evolution of compounds with short- 
distance atomic arrangement that cannot be directly analyzed by XRD. 

In certain cases it is possible to use the thermogravimetric method in a 
range of temperatures from ambient to 200°C to indirectly measure the 
specific surfaces of clays (internal and external surfaces) by impregnation 
of the sample with a monomolecular layer of an organic material (e.g. the 
EGME method) or of a gas (e.g. the BET method). 



224 



Mineralogical Analysis 



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Thermal Analysis 



225 



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226 



Mineralogical Analysis 



Instrumental thermal dilatometric methods developed by ceramists can 
be used instead of manual measurements of contraction-dilation 
(coefficient of linear extension). 



7.2 Classical Methods 



7.2.1 Thermogravimetric Analysis 



Principle 

Variations in the mass of the sample (losses or increases) are recorded as 
a function of temperature or time. For soil studies, a temperature range 
between ambient temperature and 1,100 or 1,200°C is generally 
satisfactory, but it may be necessary to study reactions up to temperatures 
of more than 2,000°C, in particular to determine melting points. 



100 



g 50 




Beginning 
of reaction 



Range of 
reaction 
(gaseous 
losses 
—►EGA) 







500 



1 End of 
reaction 



1,000 t°C 



100 








500 



1,000 t°C 



Fig. 7.2. Principle of thermogravimetric analysis m = f(t) (on the left) and com- 
parison with differential thermogravimetry dm/dt = f(t) (on the right) 



In dynamic analysis, the mass of the sample (m) is heated at a constant 
rate according to a linear programme based on temperature or time 
(Fig. 7.2). The extent of the reaction interval, the shape of the curve and 
the nature of the gases released provide information on the nature of the 
soil sample and its thermal stability. 

In TGA by derivation, the first derivative of the variations of mass is 
recorded as a function of time or of temperature. Two "static" methods 
can also be used in certain cases, but take a very long time to implement: 



Thermal Analysis 227 

- The isothermal method where the sample is subjected to a constant 
temperature and the sample weight is recorded over time until their 
equilibrium value is reached; 

- The isobar method where the sample is maintained at a constant 
pressure and the weight recorded as a function of temperature; the 
pressure can exceed 500 atm. for certain materials. 

Clay minerals contain molecules of water and hydroxides bound to the 
crystal lattice at different energies. During the rise in temperature, this 
water is gradually moved and eliminated in the form of gas. 

In the soil, interstitial water of hydration or adsorption that is not 
bound is generally released first by dehydration. Dehydration does not 
cause the destruction of the lattice but can cause a modification in the 
arrangement of the continuous poly- or monomolecular layers (internal 
and external surfaces of 2:1 clays and interlay er exchangeable ions) along 
with a contraction of the interlayer space. This phenomenon is reversible. 
Water in cavities at the base of the tetrahedrons will be more vigorously 
bound. On the other hand, the phenomenon may be irreversible in soils 
with allophane, and rehydration will not be able to reach more than 
around 10% of the initial moisture content. This is also true of hydrated 
halloysite, 4H 2 0, which gives metahalloysite, 2H 2 0. 

The hydroxyl OH" ions bound to oxygen atoms at the top of the 
tetrahedral or octahedral units, or present in the external continuous 
layers of 1:1 clays, or in the internal or external layers of 2:1 clays (or 
hydrated 1 : 1 halloysite) are then moved. Their elimination is irreversible 
and is accompanied by the destruction of the structure (dehydroxylation). 
The appearance of new forms at a higher temperature can then be 
recorded, for example kaolinite giving metakaolinite then mullite. But 
certain transformations that occur without weight loss are not detectable 
by TGA. In this case the DTA or DSC spectra have to be recorded. 

It should be noted that the transition between free H 2 and OH~ of 
hydroxyls is not always clear as the water bound to the lattice can start to 
leave before the interstitial water is completely eliminated. In this case 
thermal analysis at controlled speed of transformation can be used 
(Rouquerol 1970, 1989; Rouquerol et al. 1985, 1988) 1 . 



The method of analysis at controlled speed of transformation makes it possible 
to measure the initial state of water with more precision. In conventional 
analysis, the programme of temperature increase varies in a linear way over 
time. With controlled speed of transformation, the pressure caused by the 
departure of the gas determines the programme for the increase in temperature 
by means of a captor. Coupling with a quadripolar mass spectrometer enables 
analysis of evolved gas. When the adsorbed water with weak activation energy 
is desorbed, the rise in temperature stops until the initial pressure is restored to 
the pre-determined point. Then heating continues. This makes it easier to study 



228 Mineralogical Analysis 

Implementation 

Reagents 

- Reagents described in Chaps. 2 and 3 for destruction of organic matter 
or carbonates, homoionic saturation, etc. 

- Clays purified for standardization (samples for the reference system of 
the laboratory and/or international standards). 

- Saturated solution of magnesium nitrate (Mg(N0 3 ) 2 , 6H 2 0, 1,250 g L" 1 
at 20°C) (to equilibrate the moisture of clays - 56% of relative 
moisture at 20°C). 

- Drying agents of varying degrees of effectiveness: 

(a) Magnesium perchlorate (dehydrite), Mg(C10 4 ) 2 , molar weight mw: 
223.23; 

(b) Aluminium oxide A1 2 3 , mw: 101.94; 

(c) Silicon dioxide (Silicagel), Si0 2 , mw: 60.08; 

(d) Anhydrous calcium chloride, CaCl 2 , mw; 1 10.99; 

(e) Anhydrous calcium sulphate (anhydrite/drierite), CaS0 4 , mw: 
136.04; 

(f) Phosphorus pentoxide (phosphoric anhydride), P 2 5 , mw: 141.96. 

Equipment 

- Platinum micro-crucibles; 

- Desiccator with saturated solution of magnesium nitrate; 

- Desiccators with different drying agents; 

- Thermal balance (Fig. 7.3). 

Briefly, a thermal balance is composed of a furnace, a balance and 
different devices for regulation and data acquisition. Weighing is 
carried out continuously throughout the thermal cycle. There are two 
types of equipment: 

- Balances placed above the mobile furnace; 

- Balances placed below the mobile furnace. 

In the first, the sample nacelle is suspended on a wire; in the second, a 
vertical rod equipped with a support holds the sample. Each system has 
certain disadvantages that must be minimized to optimize measurements. 

The balance must allow all losses or increases in mass to be recorded 
as a function of the temperature and time under all experimental 



the mechanisms of hydration and possibly the short- or long-distance atomic 
structure. 



Thermal Analysis 



229 



conditions. The temperature of the furnace can reach 2,400°C and 
radiative and magnetic phenomena may occur. The quality of 
measurements must be the same as with any other analytical 
microbalance (Pansu et al. 2001). The capacity can vary considerably 
depending on the use envisaged. For soils, a choice has to be made 
between (a) relatively big samples, i.e. 100 mg to 1 g with a sensitivity of 
10~ 5 -10~ 6 g and (b) micro-samples, i.e. from 1 to 50 mg (e.g. piezo- 
electric balances with a sensitivity of KT 8 -10~ 12 g). 



Sample 




1 



Power 



Regulation! 



Program- 
ming 



Thermocouple 
Oven 



Measured 
temperature 



Weghing and 

compensation 

system 



Data 
acquisition 



Fig. 7.3. Example of a system for thermogravimetric analysis 



Different types of balances are available: deflection balances (e.g. with 
torsion, beam balance, cantilever, with a beryllium bronze spring) but 
electronic systems without a beam are rarely used because of the 
disturbances that can be caused by radiative phenomena. 

The furnace is designed as a function of the temperature. It can be the 
high frequency induction type or more generally the resistance type 
(Table 7.2). Kanthal resistances enable measurements at 1,200°C, 
rhodium resistances at 1,800°C and tungsten resistances at 2,500°C. 



The furnace is an assembly of metal and ceramic components (high 
density alumina) which allow resistances to be insulated to ensure 



230 



Mineralogical Analysis 



homogeneous temperatures in the test zone, and also to allow sealing in 
the case of controlled atmosphere. The furnace has a sophisticated 
regulation system. The speed of heating must result in a uniform 
temperature in the test zone and be monitored by thermocouples 
appropriate for the work temperature (Table 7.3). The programming of 
the heating cycle must be highly reproducible. The sample must be 
located in a precise spot in the furnace. The data acquisition system must 
enable variations in weight and temperature to be recorded 
simultaneously, and also to carry out a certain number of mathematical 
operations (e.g. first derivative, surface of the peaks), to control the 
temperature programme, and finally to store and print the data. 



Table 7.2. Type of resistances - thermal ranges 



material 


temperature 
used (°C) 


melting 

point 

(°C) 


material 


temperature 
used (°C) 


melting 

point 

(°C) 


constantan 


750 


1,200 


rhodium (Rh) 


1,800 




nichrome 


1,000 


1,500 


molybdenum 3 


2,200 


2,500 


chromel - 
alumel 


1,100 




tantale 


- 


2,850 


kanthal 


1,350 
1,400 


1,755 


tungsten b 


2,800 


3,400 


platinum 
(Pt) 








Pt-Rh 10% 


1,500 










Pt-Rh 13% 


1,700 




a Hydrogen atmosphere 




super 
kanthal 


1,600 




b Mechanical resistance up to 1,650°C 



Procedure 

The methods have to be standardised if the data obtained in different 
series is to be compared. The technical characteristics of the thermal 
balance determine certain obligatory parameters. The nature of the 



Thermal Analysis 



231 



materials to be studied determines the selection of other diagnostic 
parameters. The samples must be crushed without heating as this could 
disturb subsequent thermal analysis. 

Table 7.3. Types of thermocouples at different temperatures 



material 


temperature (°C) 


material temp. (°C) 


copper- 
constantan 3 


400 
(standard) 


tungsten - 2,200 
rhenium 


iron-constantan 


800 
(standard) 


tungsten - 20% 2,400 
rhenium tungsten 


chromel- 
constantan 


1,000 
(standard) 

1,000 
(standard) 


tantalum carbide - 3,000 
graphite b 


chromel - alumel 


a Alloy 55% copper + 45% nickel 


nickel chromium - 
nickel 


1,370 


b Argon atmosphere 


platinum-platinum 
rhodium (10% or 
13%Rh) c 


1,750 


c The thermopiles allow the 
output signal to be increased 
without amplification 



Initially rough samples (from which organic matter has been 
eliminated) are reduced by moderate wet crushing. The sample must have 
a regular particle size and, after air drying, pass through a 0.1 mm sieve. 
Samples of clays that have been purified and saturated using the methods 
described in Chap. 3 can also be used. It should be noted that too fine dry 
grinding can distort the results. 2 

Adjust the moisture of the sample by placing it in a desiccator 
containing saturated magnesium nitrate for 48 h (relative moisture should 
be 56% at 20°C at normal pressure). This treatment homogenizes the 
hydration layers of any interlay er cations that may be present. 

Weigh a given weight of sample (5-20 mg) suitable for the range and 
sensitivity of the balance. Pack the sample in a platinum crucible as 



2 Dry grinding can modify the nature of the basal faces and consequently their 
physical properties. For example, the exchange capacity and some thermal 
properties of kaolinite can be changed (collapse of the peaks) by too fine 
grinding which can result in fractures perpendicular to the basal faces and 
subsequent breakdown of layers. 



232 Mineralogical Analysis 

regularly as possible to limit differences in thermal diffusity. Place the 
sample in the thermal balance. Adjust the position of the sample to that of 
the measurement thermocouple in the furnace. 

Programme the instrumental variables with the management software, 
i.e. the linear speed of heating (e.g. 10°C min -1 ), atmosphere of the 
furnace, final temperature, etc. 

Observations 

For clay the best quantitative measurements are obtained on 
homoionically saturated samples (Na + , K + , Ca 2+ or Mg 2+ ), after 
elimination of organic matter, soluble salts, ferrous iron, etc. Homoionic 
saturation of clay enables: 
- with Na + ions, improved differentiation between adsorbed water and 

water bound to the lattice; 
-with Mg 2+ ions improved separation of 2:1 clays from 1:1 clays on the 
basis of adsorbed water. 

The presence of organic matter (OM) modifies weight loss of mineral 
origin. The loss of H 2 + C0 2 of the OM must be measured. An inert 
atmosphere can be used to mitigate this phenomenon if it is not too 
serious, or losses can be estimated by analyzing emitted gas (e.g. by 
coupled EGA-FTIR). 

Ferrous iron will oxidize during heating and increase in weight 
according to the following equations: 

Fe 2+ -> Fe 3+ + e~ 
or Fe(OH) 2 -> FeO + H 2 Ot 

2 FeO + O -> Fe 2 3 

As oxidation of ferrous iron does not result in easily measurable 
variations in mass, it may be preferable to eliminate it. The same is true 
for manganese and cobalt. An inert atmosphere can also be used. The 
elimination of soluble salts avoids secondary recombination. 

The choice of the nature of the crucible and its geometry can modify 
the results. The crucible should not react with the sample, with the 
selected atmosphere or the evolved gas. Certain metals have a catalytic 
action. Crucibles made of alumina are relatively porous, silver can be 
used for medium temperatures, platinum can be used for a temperature of 
1,500°C. The walls of the crucibles must be as thin as possible 
(approximately 0.5 mm) to minimize variations in temperature. In the 
same way, the sample layer should be as thin as possible to ensure that 
the temperature in the centre is the same as at the edges. As certain 
minerals are expansible or likely to generate projections, deeper crucibles 
are sometimes necessary or semi-permeable lids have to be used which, 
however, can modify the gas flow of the losses. 



Thermal Analysis 



233 



The speed of the increase in temperature influences the decomposition 
of the sample. For a given temperature interval, slow decomposition is 
more realistic, than too rapid decomposition which can cause 
displacement of the characteristic temperatures, a steeper decomposition 
slope, etc. For exemple, Kotra et al. (1982) showed that a siderite 
(FeC0 3 ) heated at 1°C min 1 had a range of decomposition positioned 
between 400 and 500°C. At a speed of 20°C min" 1 this range moved to 
between 480 and 610°C. However, a micro sample displays fewer 
variations than a sample of greater mass. Low speed also enables 
detection of compounds that only display weak inflection at higher 
speeds. 



100 



CO 
CO 
03 

E 
o 

CD 
D) 

c 

03 



c 

CD 
O 

i_ 

CD 

Q_ 



90 



80 



TGA t ~~^\ T t 

0.2% moisture \ 

\ Dehydroxylation 


DTG ^\ 


\ / 13.6% 

y h 2 o 

/ s 'X 




\ T 

\ 

522.6 °C 

I i I 



100 200 300 400 500 600 700 800 900 °C 



Fig. 7.4. Example of TGA and DTG curves for kaolinite (Macon, France). Mass 
of sample: 25 mg; speed of heating: 10°C min -1 ; atmosphere: 
nitrogen 30 ml_ min -1 ; and platinum crucible. 



The atmosphere of the furnace can greatly influence the results with 
respect to the nature of the decomposition products and types of 
reactions. Vacuum, inert or reactive atmosphere induce very different 
thermal spectra. It may be useful to analyze evolved gases. 



234 Mineralogical Analysis 

Differential thermogravimetric analysis (DTG) 

In this type of analysis, peaks are obtained whose surface is directly 
proportional to changes in the mass of the sample (Figs. 7.2 and 7.4), 
which facilitates quantitative analysis. When the variations in weight are 
null one obtains a return to the base line for which drn/dt = 0. The curves 
give an inflection point in the zone where the variation in mass reaches its 
maximum. The separation of overlapping phenomena is facilitated. 



Discussion 

The water contents of a clay can be measured on a sample in equilibrium 
with an atmosphere at approximately 56% relative moisture (obtained by 
a mixture of nitrate of magnesium saturated in water). In this way an 
initial point of reference can be obtained that takes into account complex 
assemblies of minerals and organominerals, and allows better 
reproducibility. 

It is possible to measure water that depends on the exchangeable 
cations. The water content can be very high for 2:1 clays of the 
montmorillonite type 3 and weak for 1:1 clays of the kaolinite type. 
However, it is not possible to measure the energy of activation necessary 
to release water with TGA or DTG. For this purpose DTA must be used 
giving the quantity of calories that generates the endothermic peak, the 
quantity of released water being identified by TGA under the same 
conditions. Existing equipment allows TGA-DTG and DTA to be used 
simultaneously in the same treatment cycle up to temperatures of 1,750°C 
or more on the same micro sample. 

Direct quantification is possible if only one reaction occurs at a given 
temperature. By insulating the components, quantification becomes 
possible for organic matter in an oxidizing medium, for carbonates (e.g. 
dolomite, aragonite, calcite, siderite), sulphides (e.g. pyrites) with very 
low contents. Here combining quantification with analysis of evolved gas 
is essential. 



3 The layers of 2:1 clays adsorb at least two layers of water molecules. They are 
disorientated with respect to one another with a tendency to form association of 
layers (turbostratic assemblies with 5 or 6 layers). 



Thermal Analysis 235 

7.2.2 Differential Thermal Analysis and Differential Scanning 
Calorimetry 



Principle ofDTA 

In DTA, the difference in temperature between a soil or clay sample and 
an inert reference material is recorded as a function of time or 
temperature with the two substances controlled by the same temperature 
control programme, at constant linear speed: 

^J- J- sample ~~ J- reference* 

This kind of analysis enables identification of the relations of 
proportionality that exist between the surface of a peak and the released 
or absorbed heat during the course of the heating programme. This heat is 
proportional to the enthalpy of reaction and thus can be used for 
thermodynamic quantification if the mass of the sample is taken into 
account. However, in DTA, the simple direct conversion of the peaks into 
unit of energy is not possible starting only from A7as a function of time. 
Indeed, AT depends on the variation in enthalpy, the calorific capacity 
and the total thermal resistance of the heat flow (R) at a given time. 
R depends on the nature and the mass of the sample, its preparation 
(compression, etc.), and the thermal surface of contact between the 
crucible and the support. These variables are temperature dependent and 
consequently have to be controlled. 

For soils, most analysis is carried out between ambient temperature 
and 1,200°C. When a reaction occurs during an increase in temperature, 
a difference in temperature is observed between the sample and the 
reference (Fig. 7.5): 

- If the temperature is lower than that of the inert reference material, an 
endothermic peak appears (AT is negative), this is the case in reactions 
of dehydration, dehydroxylation, fusion, evaporation, sublimation, etc. 

- If, on the contrary, the temperature of the sample exceeds that of the 
reference, an exothermic peak appears (AT is positive), this is the case 
for oxidation phenomena (combustion of OM, oxidation of sulphides, 
oxidation of ferrous iron, certain nucleations or decomposition with 
neoformation. 

The recorded differences in temperature are related to the change in 
enthalpy, but this does not exclude the possibility of two exothermic and 
endothermic reactions occurring simultaneously. When this is the case, 
the absence or depression of the DTA peak does not imply the absence of 
a reaction. 



236 



Mineralogical Analysis 



Contrary to TGA, DTA can produce peaks even if there is no loss or 
increase in weight, e.g. in reversible second-order transformations: 
variation in specific heat, magnetic susceptibility, Curie point or a/(3 
allotropic transformation of quartz. TGA and DTA are complementary 
techniques. 

The shape, size and temperature of the peaks are influenced by 
instrumental factors such as the speed of heating, the nature of the sample 
support and of the thermocouples. 

Small samples give a better resolution of the peaks and allow faster 
heating. Slower speed can increase sensitivity, but to the detriment of 
temperature, precision, and resolution. A dynamic atmosphere is 
preferable to a self-generated static atmosphere. This allows continuous 
evacuation of evolved gas, thus reducing the risks of artefact reactions at 
higher temperatures. These gases can then be analyzed enabling 
identification of the molecular structure of the compounds that caused the 
gaseous emission. 



Fig. 7.5. 

Diagrammatic 
output of 

differential thermal 
analysis. 
BD, peak width; 
EC, peak height; 
BCD, peak surface; 
AF, base line 



AT 



Point of 
thermal rupture 

BED 



A7=0 




A7~- 
endothermic 
peak 



Exothermic 
peak 



it 



200 400 



600 



800 



°C 



The range of different types of equipment sold by different 
manufacturers means complex mathematical demonstrations are not 
required to validate the different parameters taken into account, see for 
example Duval (1963), Watson et al. (1964), Garrels and Christ (1965), 
Allen (1966), Gray (1968), Mackenzie (1970), Brennan (1971), Miller 
et al. (1973), and McNaughton and Mortimer (1975). 

Principle of Differential Scanning Calorimetry 



Other techniques to measure energy are grouped under the name of DSC. 
DSC techniques are often badly defined because patents use the same 
term for different concepts. 



Thermal Analysis 



237 



The term DSC applies to apparatuses able to measure specific heat, or 
the heat capacity of a sample, and to quantify the energy of the reactions 
during the heat treatment. In DSC with power compensation, the sample 
and reference are continuously maintained at the same temperature by 
individual resistances. The parameter that is recorded is the quantity of 
power consumed by the compensation resistances, that is to say d(AQ)/dt 
or dH/dt in millicalories per second as a function of the temperature 
(controlled linear increase). 

When a reaction occurs in the sample, thermal energy is added or 
removed. The quantity of energy added or removed is equivalent to the 
quantity emitted or absorbed by a given transition. The recording of this 
balance of energy is a calorimetric measurement of enthalpy. 



A T= Tsample -^reference AT=r S ample-Tref 











J- 


A 








b 


_^V\A^^ 





Power 
compensation 



12 



S ^ R 



C pt 



sensors 




One resistance 



One resistance 



Two individual 
resistances 



Fig. 7.6. Thermal systems using measurements of energy: a, classical DTA; 

b, Boersma DTA with heat flow or conventional indirect DSC; and 

c, DSC with compensation of power 



In the Boersma technique, also known as DSC or DTA with heat flow, 
AT = r samp i e - r reference are measured like in DTA. The sensors are placed 
below the crucibles to reduce the effects of variations in the thermal 
resistance of the sample. This way of assembling the components is 



238 Mineralogical Analysis 

similar to other DSC heat flow equipment that records dAq/dt type data 
or, with thermopiles, dQ samplQ /d t - d0 reference /df = d(AQ)/dt enabling the 
quantitative measurement of enthalpy. 

In modulated DSC, the sample is subjected to a linear increase in 
heating (for example 10°C min" 1 ) on which a sinusoidal modulation of 
temperature (of 30 s and amplitude of 1°C) is superimposed resulting in 
a cyclic heating profile. There is thus a speed of constant subjacent 
heating and instantaneous measurements at sinusoidal speed. The 
deconvolution of the flow profile resulting from the heating profile 
provides the total heat flow as in conventional DSC, but also separates 
flow into two components: reversible specific heat and non-reversible 
kinetic heat. This technique enables improvement of resolution and 
sensitivity, because low speed favours good resolution and high heating 
speed favours good sensitivity. 

The functioning temperature of the DSC apparatuses with power 
compensation is generally limited to 750°C, which is too low to study 
certain reactions in the soil. Classical DSC makes it possible to reach 
temperatures similar to those of DTA or TGA. 

Materials 

These must be reference products and be ground to 0.1 mm in an agate 
mortar. 

- Products of known melting points to calibrate temperature (Table 7.4). 

- Inert reference: alumina (A1 2 3 ) calcined to 1,200°C, and ground to 0.1 
mm in an agate mortar does not show any effect of heating except 
possibly a few irregularities in the base line. 

- Purified clays belonging to the laboratory reference set homoionically 
saturated by Mg 2+ , Ca 2+ , or Na + . Clay samples from industrially 
exploited sedimentary deposits should be distinguished from those 
coming from soils. 

- Pure minerals from the laboratory reference set and industrial reference 
products for analysis. 

- 1,250 g (Mg(N0 3 )2,6H 2 0) L" 1 of magnesium nitrate saturated aqueous 
solution at 20°C. 

Equipment 

- DTA micro crucibles made of platinum (or possibly alumina, quartz, 
tungsten, zirconium oxide, nickel, or aluminium). 

- Desiccator with Mg(N0 3 ) 2 , 6H 2 saturated solution. 

- Desiccator with desiccating agents. 



Thermal Analysis 



239 



Analytical balance (10~ 6 -10~ 8 g) with a weighing range of 1-100 mg 
for quantitative analysis. 

Apparatuses: industrial DTA-DSC instruments are designed and built 
according to different concepts, and this can make comparison of data 
difficult. It is advisable to use a laboratory reference set to ensure the 
precision and relevance of the results. The equipment generally 
comprises different components (Figs. 7.6 and 7.7): 

Table 7.4. Calibration products for thermal analysis 



product 




formula 


melting point 
(°C) 


mw 
(g mo!" 1 ) 


naphthalene 


C 10 H 8 


CO 


80.2 


128.16 


phenanthrene 


C 14 H 10 


tfb 


99.3 


178.22 


benzoic acid 


C7H6O2 


P y — cooh 


122.3 (sublimation) 


122.12 


barium chloride 


BaCI 2) 2H 2 


130.0 (-2 H 2 0) 


244.31 


a calcium sulphate 


CaS0 4) 


2H 2 


180.0 (-2 H 2 0) 


172.18 


tin 


Sn 




231.97 


118.70 


cadmium carbonate 


CdC0 3 




350.00 


172.42 


zinc 


Zn 




419.4 (ignition at air 
contact) 


65.38 


lithium bromide 


LiBr,H 2 ( 


D (deliquescent) 


553 


104.87 


quartz 


Si0 2 




573 


60.06 


aluminium 


Al 




660.2 


26.98 


a calcium carbonate 
(468calg~ 1 ) 


CaC0 3 




850 (C0 2 + CaO) 


100.09 


silver 


Ag 




960.8 


107.88 



Standard material used to measure heat of reaction. 



A furnace equipped with a device to programme temperature, to 

control atmosphere and to accelerate cooling (between 20 and 40 

min before a new cycle starts); 

A support for the sample equipped with differential temperature 

detectors; 

A management station for analytical programmes and recording of 

measurements. 



240 



Mineralogical Analysis 



Temperatures exceeding 2,000°C are required for ceramics. For soils, 
a temperature of 1,200°C is generally sufficient but 1,600°C may be 
needed. 

The position of the sample support and the inert reference can vary 
considerably (Fig. 7.7). The thermocouples can be placed in or outside 
the crucibles (or to even welded into a cavity in the crucible). Metal 
crucibles give smaller peaks than ceramics with a faster heat transfer thus 
limiting the risks of deformation of the peaks. For studies up to 
approximately 1,000°C, chromel-alumel thermocouples generate a 
significant electromotive force (EMF = 45.16 mV at 1,100°C) but 
relatively low chemical resistance. On the other hand, platinum-rhodium 
or platinum thermocouples generate EMF = 10.74 mV which requires 
amplification, but can reach temperatures of 1,500°C and are chemically 
much more resistant. 



Reference Sample 



Fig. 7.7. DTA 

equipment 




V 



Power 



Regulation 



Thermocouple 
Oven 



Data 
acquisition 
and control 



AT 



7°(C) 



Amplifier 



The position of the thermocouples in the sample and in the reference 
must be perfectly symmetrical to avoid any variation in the shape of the 
peaks or displacement of the base line. The measurement station should 
include a micro computer and associated peripherals and ensure: 

- Complete piloting of the temperature cycles (speed of heating, 1-50°C 
min" 1 with stabilized voltage, temperature control by means of 
thermocouples, carrying out instructions, etc.; 

- Control of the atmosphere (purging circuits, admission of carrier gas, 
admission of inert or reactive gases, EGA output, regulation of flows 
and pressure); 



Thermal Analysis 241 

- Simultaneous acquisition of data on temperature and heat transfers with 
very weak inertia, thermodynamic calculations and print-outs of the 
results. 
As is true for TGA, standardization of the procedures is essential. 

Sample 

Use a sample of whole soil ground to 0.1 mm (should not be over- 
ground). Organic matter in humic soils produces a strong exothermic 
reaction culminating at 300°C (573 K) which obstructs the endothermic 
peaks of dehydration towards 250-400°C if analysis is carried out in air 
or in an oxidizing atmosphere. It is consequently better to destroy OM 
using the procedures described in Chaps. 2 and 3 (or to extract it and 
use DTA). On whole soil, the peaks are generally of low intensity. Quartz 
can obstruct, but its peak at 573°C can be used as a marker. 

To improve sensitivity, it may be better to use isolated purified and 
enriched fractions of a given particle size, homoionically saturated in the 
case of clays and, after air drying, stored in a solution saturated with 
Mg(N0 3 ) 2 ,6H 2 to maintain constant relative moisture (the size and 
crystallinity of the particles influence the temperature, size, and shape of 
the peaks). 

In sandy fractions (2.0-0.05 mm) primary minerals and concretions 
rich in iron and manganese can be measured. The endothermic peak of 
quartz is generally observed at 573°C. 

Silt fractions (0.05-0.005 mm) often represent a more complex 
medium: they contain both primary minerals and deteriorated forms and 
possibly forms of clay minerals > 2 |Lim. 

Coarse or fine clays (< 2 |Lim) often give much more intense and 
quantifiable peaks than whole soils. Substances with short-distance 
arrangement (e.g. allophane, ferrihydrites) are found in this fraction. 
Homoionic saturation by a cation (Na + , K + , Ca 2+ , Mg 2+ , Al 3+ ) enables 
certain properties such as levels of hydration to be revealed. The first 
endothermic reaction of volatilization of the adsorbed water varies with 
the number of water molecules associated with the cation. The cations 
affect the size and the shape of the peaks, generally without significantly 
modifying the temperature at which they appear. 

It is possible to mix the clay fraction with alumina to avoid caking, 
retraction, and cracking of the sample, but this has a diluting effect. 



242 Mineralogical Analysis 

Two clay samples from the same profile can be compared under the 
same experimental conditions. The reference sample will be then one of 
the samples. If they are identical, AT will be equal to and there will be a 
base line with no peak, as the two samples will cause the same 
phenomenon to occur at the same time. 

Reference Material 

The standard must be thermically inert in the temperature range to be 
used. It must have a thermal conductivity or a thermodiffusivity close to 
that of the sample. The particle size should be that of soil (ground to 0.1 
mm) which allows close contact with the crucibles as well as a favourable 
bulk density (same proportion of pores near the ground soil). 

Generally alumina is used (A1 2 3 calcined at 1,200°C for 1 h). This 
reference can be used for several measurements, but slight hygroscopicity 
of the product may be observed after 4 or 5 measurements. 

A clay burnt at 1,200°C and then ground can also be used, but 
problems often occur with respect to the base line. For example between 
rough kaolinite and kaolinite calcined to mullite at 1,200°C, thermal 
conductivity will vary by a factor of two and certain reversible reactions 
may occur. Quartz, magnesia, or aluminium sheets are sometimes used as 
reference materials. 

Procedure 

The procedure is similar to classical qualitative or quantitative DTA- 
DSC analysis, and takes into account the factors mentioned earlier. 

- Weigh on the laboratory balance (± 1/100 mg) a given mass of sample 
(1-100 mg), suited to the characteristics of the apparatus and an 
identical quantity of inert reference; for DSC with power compensation, 
an empty crucible similar to that used for the sample can be used as 
inert reference. 

- Pack as homogeneously as possible in platinum crucibles. 

- Find the optimal position for the crucibles (in suitable supports) with 
respect to the thermocouples and the most homogeneous zone of the 
furnace. 



Thermal Analysis 243 

- Cover the crucibles with a lid if required (the displacement of evolved 
gas will be modified). 

- Regulate the speed of linear increase in temperature; higher speed can 
increase some peak temperatures, but can also improve sensitivity (peak 
height); generally the range is between 5 and 20°C min 1 but certain 
materials allow speeds of 0.1-200°C min 1 (it is also possible to use 
the reverse technique with recording during cooling). 

-Adjust the atmosphere of the furnace taking into account possible 
analysis of the gaseous phase by interfacing with EGA. 
Software packages can monitor and control operating conditions 

including calculations and printing out the results, in this case all the 

parameters are optimized. 

Interpretation of the Results 

Table 7.5. Main thermal effects on the clays of soils 



range of 

temperature signals 

(°C) 

50-250 endothermic peaks 

loss of absorbed water, interlayer water (e.g. allophanes, 
halloysite, smectite) 

400-700 exothermic and endothermic peaks 

crystallisation reactions (e.g. gels of iron or aluminium oxides) 
diagnostic peaks of the crystalline forms 
reactions of dehydroxylation 

900-1050 exothermic peaks 

reaction of nucleation or recrystallization (e.g. kaolinite- 
chlorites) 



The same procedure is used for rough samples and extracted fractions 
of different degrees of purification under inert or reactive atmosphere 
depending on the nature of the sample studied. 

"Pure" standard minerals or minerals in known mixtures (90:10, 
80:20%, etc.) can be treated to trace the calibration lines with the same 
criteria thus enabling quantification of the samples (a reference set of 
laboratory minerals is necessary). 

A distinction must be made between clay minerals from sedimentary 
deposits whose peaks are generally well defined and minerals from 
different soils after purification and concentration. 



244 Mineralogical Analysis 

First, well-defined peaks are identified (e.g. the endothermic peak at 
500°C and the exothermic peak towards 900°C for kaolinite). The shape 
of the peaks is an indicator of the thermal process: fusion results in a 
narrow endothermic peak, decomposition often produce a broad 
endothermic peak which is often asymmetrical, a second-order transition 
results in only a slight inflection. Certain minerals give well-defined 
peaks that are used as markers of characteristic transitions in the thermal 
programme. 

The slope of the base line and the magnitude of the peaks should be 
taken into account. The main thermal transitions observed in clays are 
listed in Table 7.5. The "low temperature" zone provides information on 
adsorbed water and on the presence of certain clays and amorphous 
substances. The medium temperature zone shows peaks resulting from 
the predominant influence of crystallinity. Finally the high temperature 
zone shows phenomena such as nucleation or recrystallization of clays. 

Certain standards are used in quantitative analysis like CaS0 4 ,2H 2 to 
enable calculation in millicalories per unit of area (calibration between a 
peak surface and a known emitted or absorbed heat). 

Each type of structure can be identified by a characteristic thermal 
curve (Fig. 7.8). For clays, purification is necessary to limit the artifacts 
caused by impurities which are likely to be superimposed in the same 
temperature zone. 

Clays with a tetrahedral layer and an octahedral layer will display 
phenomena related to the decomposition of the octahedral layer. The 
exothermic volatilization of hygroscopic water may be accompanied by a 
recombination of the elements of the tetrahedral and octahedral layers. 

Quantitative DTA is possible for clays presenting endothermic peaks 
associated with loss of water bound to the crystal. This is the case of 1 : 1 
clays like kaolinite, but 2:1 clays like montmorillonite, vermiculite, or 
illite are difficult to quantify with this method. 

Deferrification reinforces the exothermic peaks of kaolinite, and iron 
inhibits exothermic phenomena. The presence of carbonate, organic 
matter, and alkaline ions must be taken into account. Hydroxides initially 
present loss of water, and then contraction linked to decomposition of the 
lattice. 

The heights of peaks are faster to use than the surfaces of peaks and 
can thus be used for approximations. Thermodynamic calculations use all 
the spectral information as well as the quantities of evolved water 
measured in TGA. 



Thermal Analysis 



245 




500 1000X o 



500 1000X 



500 1000°C 



Fig. 7.8. Examples of DTA thermal diagrams on soil minerals (J Gautheyrou, 
set of reference standards from IRD Bondy, France, unpublished data) 

In DSC, the curves are proportional to the enthalpy: 
peak surface = m AH/k 

AH; heat of transition; m; reactive mass; k; coefficient of calibration 
(independent of the temperature measured by DSC with power 
compensation). 



246 



Mineralogical Analysis 



7.3 Multi-component Apparatuses for Thermal Analysis 



7.3.1 Concepts 



Balance 




Balance 



Neutral f 
gas 



Oven 



Reactive 



Balance 



N eutral f 
gas 



Oven 



gas 



Liquid 
nitrogen 
trap * 
GPC or FTIR 
coupling 

Vacuum^" 

Reactive 
gas 



MS 
coupling 



Fig. 7.9. Example of gas sweeping devices for TGA or TGA-DTA. (a) Simple 
displacement by a neutral carrier gas crossing the balance and 
protecting it from corrosion; (b) system with two gases - one neutral 
and one reactive - with horizontal sweeping of the sample; and 
(c) system with purging by vacuum and later sweeping by neutral gas 
or neutral gas + reactive gas 

The development of new apparatuses was based on several different 
concepts. In multi-component equipment, several dedicated apparatuses 
are run by a management station and measurement is computerized. This 
type of multi-function equipment enables multiple measurements on the 
same sample subjected to a thermal profile with controlled atmosphere 
and pressure. Suitable sensors can measure TGA, DTG, DTA and DSC 
simultaneously thus reducing the time required for analysis, increasing 
precision and allowing more thorough characterization. 

Automated introduction of the samples in the furnace is useful in the 
case of equipment with a cooling cycle of less than 30 min (return to 
ambient temperature after heating to 1,700°C). 



Thermal Analysis 247 

Certain dedicated measurement peripherals can also detect or quantify 
evolved gas. This quantification is essential to complete certain DTA 
spectra (e.g. simultaneous exothermic and endothermic reactions). 

The methods selected can be programmed and used in sequence, 
providing a powerful reference frame, e.g. for the identification of clays 
and the study of transformations in structure, recrystallization, forms of 
water, thermal stability or thermal oxidation. 



7.3.2 Coupling Thermal Analysis and Evolved Gas Analysis 

The analysis of emitted or emanating gas may require coupling of 
equipment of varying degrees of complexity. A small furnace with an 
open configuration should be used that is able to ensure rapid evacuation 
of decomposition gases (Fig. 7.9). The circulation of a carrier gas and the 
possible presence of reactive gases causes changes in pressure and 
convection phenomena linked to pressure and local temperatures (as well 
as thermomolecular forces able to create heterogeneous temperatures 
inside the furnace) that are controlled by a system of screens. Below is an 
overview of the most widely used systems. 

Simple detection of evolved gas (EGD): a neutral carrier gas draws 
evolved gas into a catharometer detector; the output signal is proportional 
to the concentration of evolved gas in the carrier gas. The nature of the 
evolved gases is not identified. It is possible to trap the heavy products in 
liquid nitrogen, and then to subject these products to further analysis, for 
example controlled pyrolisis by thermal analysis. 

Analysis of evolved gas (EGA) can be performed by coupling 
equipment of varying complexity. Gas chromatography, coupled with 
simultaneous TGA-DTA equipment, makes it possible to characterize 
evolved gas by automatic discontinuous injections (it is also possible to 
trap the fractions that are not analyzed in liquid nitrogen). Integration of 
the peaks makes it possible to quantify evolved and separated gas as a 
function of their retention times. 

Coupling a Fourier transform infra-red spectrometer (FTIR) enables 
analysis of time of flight thanks to the speed of acquisition and the 
sensitivity of FTIR. A flow of nitrogen automatically transfers the 
evolved gases towards the spectrometer. Resolution is about 4 cm" 1 . Each 
temperature point is stored in a numerical file corresponding to the 
number of the selected wavelength at this point. Water is identified in the 
3,600 and 1,600 cm" 1 zones, CO and C0 2 in the 2,000 to 2,400 cm" 1 
zones. 



248 



Mineralogical Analysis 



> 






rmrx 










1 


8^ 


— h. U 1 U 


7 "* C~ 


V 







1 


CD 


\ 


V TGA 




Jgk kih'A 


— 


J 


X 




\ 




i -*H }543.0 







^ . 




",*227.0 DTA 


m.7\\ i 




■J 


■ 


' 


99.1 °C\ , 




•lV 


\\ 


i 






2* 


* i 
1 \ 


>: 
*'.' 


i^ ^ — — 




i 


■ 




3< 


\\ 


J [ 

i 


1 1 






■ 


-20 


- 


\ ', 


!j 


V i; 








• 


,' 


u 


Y» | 


■ 




4. 




i 


® 


y90.8 










.; 1 




A* 












5. 




L 1 




3 




-40 






i 




1 














' ' 100 200 ' 'M ' 4( 


iO 5tlu 600 700 800 900 


t°C 




I 


i 


fi 








£ 


I 


i 








w 

c 

B 




i 


CO W 1/20, 


-* CO, 






S. 


i 

f 

1 
i 

1 

1 

1 
1 

; 


i 

i 
1 

1 


1 1 

;i 

i i 
p i 

i i 

,' i 

i i 

! 


A 

i ! 
/ i 

/ 
/ 








y 


\ 


* 


1 








-m/e = 18 


\ 

«** ^ ^_ 


j r 


1 


i 

■ 

J. 






m/e = 44 




J l 


/ 


i 

v^ iw 

















5 

w 

o 
to 

CO 



Fig. 7.10. Decomposition of the standard calcium oxalate (CaC20 4 , H 2 0) 
studied by coupling TGA, DTG and DTA (at the top) and mass 
spectrometry (at the bottom) atmosphere, air; speed of heating, 5°C 
min -1 , sample mass, 50 mg. 

1. CaC20 4) H 2 -> CaC 2 4 + H 2 OT (12.2% H 2 0) 

2. CaC 2 4 -> CaC0 3 + COT (19.1% CO) 

3. CaC0 3 -^CaO + C0 2 T (30.1% C0 2 ) 



Thermal Analysis 249 

Coupling with a mass spectrometer is very powerful, but difficult to 
implement, as the mass spectrometer operates under a vacuum of about 
10~ 5 -10~ 6 mbar. Interfacing with the thermal analyzer is achieved by 
means of double stage separators. A quadripole mass spectrometer 
enables rapid scanning in the mass range of evolved gas up to 
approximately 100, including 12 C, 16 CH 4 , 28 CO, 44 C0 2 , 18 HOH, 16 0, 32 2 , 
34 H 2 S, 64 S0 2 , 80 SO 3 . This range is not sufficient for the observation of 
organic materials resulting from controlled pyro lysis (cf. Chap. 12) as 
the molecular weights are distributed over a larger range, up to 500 or more. 

Figure 7.10 shows analysis of the thermal decomposition of oxalate of 
calcium, which can be used as a standard for calibration. With a 
combined mass spectrometer with a relatively low speed of acquisition 
and air atmosphere, decomposition reaction 2, which gives calcium 
carbonate and carbon monoxide, gives only one peak at mass 44 (C0 2 ). 
With nitrogen atmosphere, formation of a CO-C0 2 mixture is observed, 
showing the beginning of decomposition of the newly formed carbonate. 
Simultaneous detection of CO and C0 2 can also be achieved by coupling 
the thermal analyzer with a Fourier transform infra-red spectrometer. 

The analysis of radioactive gases emanating from rocks in their solid 
state at different temperatures can be performed by coupling thermal 
analysis with a detector of a-particles. 

References 

Allen JA (1966) Energy Changes in Chemistry., Allyn-Bacon Newton, MA 
Braudeau E (1987) Mesure automatique de la retraction d'echantillons de sol 

non remanies. Science du Sol, 25, 85-93 
Braudeau E (1988) Equation generalisee des courbes de retrait d'echantillons de 

sols structures. Comptes rendus acad. Sci. Fr., serie 2, 307, 1731-1734 
Braudeau E, Costantini JM, Bellier G and Colleuille H (1999) New device and 

method for soil shrinkage curve measurement and characterization. Soil 

Sci. Soc. Am. J., 63, 525-535 
Braudeau E, Frangi JP and Mothar RH (2004) Characterizing non-rigid dual 

porosity structured soil medium using its characteristic shrinkage curve. 

Soil Sci. Soc. Am. J., 68, 359-370 



250 Mineralogical Analysis 

Duval C (1963) Inorganic Thermo gravimetric Analysis., Elsevier. Amsterdam. 

2eme edition 
Garrels RM and Christ CL (1965) Solutions, Minerals and Equilibra., Harper- 
Row New York 
Gray AP (1968) Symposium Analytical Chlorimetry, Porter R.S. and Johnson JF 

ed., Proc. Am. Chem. Soc, Plenum New York, 209 
Kotra RK, Gibson EK and Urbancic MA (1982) Icarus., 51, 593 
Mackensie RC (1970) Differential Thermal Analysis . Academic London. Vol. 1. 
McNaughton JL and Mortimer CT (1975) La calorimetrie differentielle a 

balayage. Butterworth, Physical Chemistry., Serie 2, 10. 43 pages 
Miller B, Giuham JM, Brennan WP, Nentzer C and Whitwell JC (1973) 

Thermochim. Acta, 5, 257 
Pansu M, Gautheyrou J and Loyer JY (2001) - Soil analysis - sampling, 

Instrumentation and Quality Control, Balkema Amsterdam, Lisse, 

Abington, Exton, Tokyo, 512 p 
Rouquerol J (1970) L'analyse thermique de decomposition construite. J. Therm. 

Anal, 2, 123-140 
Rouquerol J (1989) Reciprocal thermal analysis. The hidden face of the thermal 

analysis. Thermodyn. Acta 
Rouquerol F, Rouquerol J, Thevand G and Triaca M. (1985) Desorption of 

chemisorbed species: its study by controlled rate thermal analysis. Surf 

Sci., 239-244 
Rouquerol J, Rouquerol F, Grillet Y and Ward RJ (1988) A critical assessment 

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calorimetry. In Characterization of Porous Solid, Kunger KK ed., 

Elsevier Amsterdam, 67-75 
Watson ES, O'neill MJ, Justin J and Brenner N (1964) DSC. Anal. Chem., 36, 

1233 



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Thermal Analysis 251 

Dunn JG (1980) L'analyse thermique, une technique de centrale de qualite dans 

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8 



Microscopic Analysis 



8.1 Introduction 

The study of the processes of genesis and weathering of the soils and the 
characterization of minerals require observations at different spatial and 
temporal scales. Pedon, horizon, macro- and micro-samples are used as a 
basis for a range of examinations in which microscopy techniques enable 
observation of the interfaces of the different phases and even of the 
ultimate stages of molecular assemblies. Complementary analytical 
probes enable in particular determination of the chemical nature (e.g. 
energy dispersive X-ray, EDX or wavelength dispersive X-ray, WDX 
analysis) and the structural organization (electron micro-diffraction) of 
these phases. 

Whereas classical wet chemical analyses provide information on 
overall evolution, microscope analyses enable identification of hyperfine 
chemical heterogeneity and facilitate understanding of transformation 
mechanisms. Qualitative and quantitative information is obtained on the 
texture of individual particles, e.g. shape, the presence of cements, 
inclusions, pores. 

Detailed morphological analysis (spatial relations of the different 
phases, preferential orientation, etc.) is performed by coupling a 
microscope to an image or texture analyser, and analysing thin sections to 
reveal the cartography of chemical segregation, micro-profiles, and to 
improve our knowledge of pedogenesis. 

The crystalline structure can be identified by changing scale: i.e. by 
studying the elementary mesh of clays, regular sequences of silicates in 
layers, modifications of the basal interlayer, the appearance of 
aperiodisms, defects, micro-cleavages, growth steps, gels with short- 
distance atomic arrangements near source crystals which bring about the 
transformation of one mineral into another at the nano-structural scale. 
Electron micro-diffraction, which is available on the majority of electron 



254 Mineralogical Analysis 

microscopes, enables identification of crystalline structures. All these 
options make microscopy an extremely powerful tool. 

Optic and electronic microscopy and the accompanying peripherals 
used to determine chemical distribution within the sample are widely used 
in pedology, mineralogy, crystallography, petrography, metallography, 
and clay geology, and provide precise information on the mechanical 
properties, localization of surface charges and exchange properties of the 
soils. 

However, it is extremely important to think about the validity of the 
observations and to adapt the scale of measurement to the exact 
requirements of the sample. Sampling conditions and the different stages 
of sample preparation should always be specified. The season should also 
be taken into account (samples taken in a wet or dry season present very 
different pore spaces in soils rich in 2:1 clay). Not only pedogenesis and 
geostatistics but also geomorphology must be used (the samples may 
require complementary sampling of the evolution of a profile, or 
sampling at a larger scale to account for the homogeneity of a zone). 
When the observations are made, it is important to be aware of the 
physical and chemical processes that can modify a sample and lead to 
erroneous results (e.g. contamination, oxidation, neoformation). It is also 
important to explore the full potential of the equipment, as the full 
capacity of apparatuses is frequently underexploited. 

Electron beams can damage the surface of materials. The depth of 
penetration of radiation may be limited to near the surface: i.e. 
approximately 1-3 nm, or on the contrary, may gradually erode the 
surface resulting in a concentration of profiles (e.g. in plasma 
bombardment, secondary ion mass spectrometry). 

As is true for all methods, the calibration of measurements is essential 
and requires a reference set of sample observations that will allow 
comparison with known and clearly identified situations. 



8.2 Preparation of the Samples 



8.2.1 Interest 

Visual examination, followed by examination with a magnifying glass is 
not enough as it only enables a rough estimate of modes of assembly and 
physical properties. This type of examination is consequently always 
supplemented by tactile examinations and other sensorial and chemical 
tests (described in Pansu et al. 2001). 



Microscopic Analysis 255 

Observations can be made under an optical or electron microscope on 
soil samples that have undergone treatments such as: 
-purification, concentration, quantitative separation into classes of 

particle size 

- separation in heavy liquids of increasing densities, up to d = 4.28 at 
20°C (e.g. bromoforme, tetrabromomethane, di-iodomethane, Clerici 
solution) 

- magnetic separation in a Frantz magnetic separator (or similar). 

In this case, individual particles are measured (shape, nature, relative 
proportions of minerals, effects and nature of weathering). To obtain 
precise information on assemblies, thin sections have to be prepared on 
glass slides making it possible to study soil transformation and to link this 
information with field observations. 

28 x 48 mm petrographic slides are the most widely used, but 1 10 x 76 
mm or even 200 x 180 mm Mammoth slides are also useful for micro- 
pedological and micro-morphological studies (however, these are very 
delicate operations). 



8.2.2 Coating and Impregnation, Thin Sections 



Principle 

The term coating is generally used in the case of massive compact 
samples with simple geometry, such as pieces of hard stone which can be 
embedded in a resin that hardens rapidly and facilitates preparation for 
analysis. 

The purpose of impregnation with resin is to consolidate soft porous 
rocks, products of rock deterioration and soils (Delvigne 1998) for which 
cohesion is required. Wet soils must be dried in a way that avoids 
modifying the structure of the sample by contraction or the appearance of 
cracks due to shrinkage. One way to limit this phenomenon in soils rich 
in 2:1 clays with low porosity is to saturate the sample with sodium 
chloride before desiccation by freeze-drying. 

For soils rich in allophanes with a water storage capacity reaching 
250% of dry soil, water can gradually be replaced by acetone, or dioxane. 
This technique is also applicable on clay soils, but in this case, samples 
treated with acetone are more fragile than those treated with dioxane 
(Tessier 1985). Acetone and dioxane are compatible with certain resins 
used for impregnation. 

Freeze-drying generally preserves the features of the sample better 
than oven or air drying. For small samples (approximately 30 cm 3 ) the 



256 Mineralogical Analysis 

use of a dehydration apparatus at C0 2 critical point gives excellent 
results. For bulky wet samples, the use of epoxy resins that are soluble in 
water is one possible solution (Moran et al. 1989). 

Preparing the thin section slides manually is very time consuming and 
considerable technical skill is needed to obtain thin sections of quality 
with the required degree of transparency and a uniform thickness of 
between 20 and 30 |Lim. Automation of coating and impregnation, slicing, 
and polishing is possible, but requires a high initial investment which can 
only be amortized by at least 60 coatings or 40 impregnations per week, 
i.e. approximately 2,000 slides of format 28 x 48 mm per year. Some 
computer-controlled modular systems enable automated production of 
super-thin sections 10 |Lim thick, with 0.25 |Lim polishing if required. 
Standard slides are 30 |Lim thick with approximately 1 |Lim polishing. (Fig. 
8.1) 

It is often preferable to have the thin section slides made in specially 
equipped laboratories because of the need for explosion-proof electric 
circuits, vapour evacuation circuits with solvent traps, plus the cost of 
maintenance of the equipment and the instability of the resins. 

Equipment 

- Laboratory equipped with a fume hood (with non-deflagrating electric 
apparatuses) 

- combined system for grinding and polishing with a disc 250 mm in 
diameter 

- cutting saw with a diamond grinding stone 350 mm in diameter 

- equipment for cold impregnation and coating under vacuum: desiccator 
300 mm in diameter with a vacuum stopcock and a device for 
admission of the mixture of resin + catalyst 

- vacuum pump with pressure gauge (0-750 mm Hg) 

- ultrasound tank 

- drying oven with ventilation (up to 100°C) 

- stylograver with a tungsten carbide point 

- petrographic glass slides, size 28 x 48 mm, 1.2 mm thickness 

- glass sheets 0.13 mm thick for use as slide covers 

- boxes for thin section slides 

- ultramicrotome with thermally regulated system of displacement 

- freeze dryer 

- vacuum desiccators with drying products such as silica gel 

- binocular microscope 

- small equipment such as glass slab, aluminium sheet, grips 

- refrigerator (to store resins) 



Microscopic Analysis 



257 



J Sawing | 



Unatsturbed 
soli sample 



Hat preceding 
Cold impregnation 



J 



<> 



? 



Glass slid© 
30x45x1 jf6 mm 



I F "l* 

\ - x rr-v* 1 .15 



Scmpte-sflde 
cementing 



™- 03 mm 



Cutting 



60 Mm prepoUshing 
30 h™ grindng with 
boron carbide 



Uncovered 
transection 

J 

End of polishing 
1 or 0,25 \im 
Thickness 20 pm 
+ 5 urn adhesive 



[Jrfiv 




Fig. 8.1. (a) Preparation of thin section slides (equipment and materials required: 
resins, press, coating, UV polymerization apparatus, grinding stone 
cutting saw, grinding polishing machine) (b) Computer controlled 
station for automated modular preparation (Struers) 



Materials and Reagents 

- Silicon carbide sheets (Carborundum-SiC) MOHS hardness: 9 

- boron carbide sheets (B 4 C) MOHS hardness: 9 

- corundum sheets (Alumina - A1 2 3 ) MOHS hardness: 9 

- diamond sheets (range of particle sizes) 

- polishing cloth 

- diamond water-ethanol soluble pastes and sprays: 3, 1, 0.25 |Lim 

- cold epoxy resins + hardener + catalyst + thinner 

- acrylic resins with low shrinkage rate 

- cold polyester resins 

- dyestuff for resin 



258 Mineralogical Analysis 

- Canada balsam (+xylene) 

- lubricants and solvents: glycerol HOCH 2 -CH(OH)CH 2 OH, oil, ethanol, 
O-xylene C 6 H 4 (CH 3 )2, styrene C 6 H 5 CH=CH 2 , 1-4 dioxane, acetone, 
99% diethylene triamine H2N-CH2CH2NH-CH2CH2NH2 

- silicone grease for grinding 

The choice of a resin will depend on its: 

- refraction index (near 1.54) 

- solubility in an organic solvent or water 

- low level of contraction and low viscosity 

- polymerization conditions 

- hardness and strength at the required temperature particularly for 
observations and quantifications by electronic microscopy of the 
SEM and EDX type. 

Procedure 

Compact Hard Samples (Rocks) 

Petrographic techniques are described in detail by Hartshorne and Stuart 

(1970). 

The sample is suitably oriented and sawn on one face, then gradually 
polished and finally stuck on a petrographic slide of format 28 x 48 mm 
(1 in. x 2 in.). After sawing off a section 2-3 mm thick parallel to the 
stuck face, thin to 30 |Lim. Polish the thin section to 1 |Lim. The thin 
section slide can be protected from abrasion and oxidation by being stuck 
onto a thin glass sheet with Canada balsam. The slide should not be 
covered if subsequent tests require specific dyes, chemistry (elimination 
of carbonates) or samples are required for SEM-EDX measurements. 

Frangible, Porous or Fissured Samples 

Samples removed in the field with their natural moisture using cylinder or 
monolith methods of soil analysis (Pansu et al. 2001) are often oriented 
vertically in the profile or possibly oriented according to the field slope 
(Fig. 8.2), these should be stored in airtight boxes then used to produce 
the 28 x 48 mm standard or Mammoth slides. 

Whenever the block is being cut during preparation, the direction of 
the field micro-section should be taken into account (Fig. 8.2). The 
sample is dried by freeze-drying or by replacing the water with acetone or 
dioxane. Slight contraction will allow the sample to be released from the 
mould unless the system of sampling with two half-cylinders is used. 



Microscopic Analysis 



259 



Directed bi- 
section 



Fig. 8.2. Pedological micro-section 




Field 
slope 



Bottom 



The block should be cut into a rough square with a cutter to reduce its 
thickness and, depending on the chosen orientation, deposited in a semi- 
flexible plastic mould (in practice, the bottom of the mould corresponds 
to the bottom of the profile). 

Impregnation 

Place the block of soil with its mould in a vacuum desiccator with a 

ground lid lubricated with silicone grease. Put the desiccator under 

vacuum at approximately 60 cm Hg (or more) depending on the boiling 

point of the solvent and the resins. The soil should be degassed for 30 

min. 

In a flexible plastic container, quickly mix the resin with the hardener 
(in the proportions recommended by the manufacturer) and the same 
volume of solvent to fluidify the mixture. Pour the resin mixture into 
the funnel at the top of the desiccator. Introduce the mixture gradually 
while monitoring vacuum, input flow and the rise in the level of the 
impregnation liquid until the sample is completely covered (plus one cm 
to allow for subsequent retraction of the resin). The mixture must also 
flow around the sample, and impregnation should take place upwards by 
capillarity. Depending on the porosity and size of the sample, 5-8 hours 
are often needed for 48 x 28 mm slides. 

Break the vacuum with care and transfer the sample under a fume 
hood. The thinner will take approximately one week to evaporate. The 
product becomes increasingly viscous, and then starts to harden (this will 
take about a month depending on the resin and dilution). Put it in a 
ventilated drying oven at 45 °C for 2 or 3 days until the sample is no 
longer sticky. Unmould it respecting the orientation. 

Cutting 

Place the block on a bed of coloured resin (approximately 2 mm thick) 

that hardens rapidly in order to be able to identify the bottom of the soil 



260 Mineralogical Analysis 

profile. Saw the hardened impregnated block according to the preferential 
orientation (a slight notch can be cut in the side of the block to identify 
the direction of the field slope). 

The circular saw should be lubricated with a solvent that cannot 
dissolve soluble salts and should be compatible with the resin and with 
any chemical analyses to be performed later on. When impregnation 
appears to be complete, cut the block into parallel sections 5-6 mm thick. 
Each section should match the format of the petrographic slides. Rapidly 
check the quality of the surface for impregnation defects like cracks or 
flatness. The surface may need to be impregnated again after cleaning 
with alcohol and compressed air. Using a spatula, coat the section with a 
mixture of resin plus catalyst and place it on a flat glass block; cover it 
with a thin aluminium film pressing to exclude any air bubbles. After 
solidification, uncover the sample, trim it with a scalpel and leave it to 
harden in a ventilated drying oven at 45°C. The section is then ready for 
polishing. 

Using a silicon carbide abrasive disc with a rather coarse particle size, 
polish until almost complete abrasion of the re-impregnation layer, then, 
after cleaning with a blast of compressed air, continue polishing with 
a sequence of diamond abrasive paste of increasing smoothness (6, 3 and 
1 |Lim). The surface must be perfectly smooth with no abrasions and no 
residues of the re-impregnation film. The material will look dull on the 
polished resin. 

Sticking the Sample on a 48 x 28 mm Petrographic Slide 
Clean the slide with solvent and dry. Write the slide number with India 
ink. Mix the resin used to fix the sample with hardener (at 60°C or cold 
depending on the nature of the sample and of the resin). With a spatula 
apply the resin to one surface of the holder slide and to the polished 
section of sample, and then rotate the two faces slowly one against the 
other moving them very gently to push out any excess adhesive and 
eliminate air bubbles. Allow to harden and trim the edges. After 48 hours, 
smooth the external face of the sample by sawing it to approximately 300 
|Lim thickness, then by polishing it with set of diamond powders of 
decreasing particle size until reaching 30 |Lim (the usual thickness of thin 
slides for optical observation gives an orange birefringence in quartz). 
Each time the particle size of the diamond paste is changed, carefully 
clean the slide to eliminate all coarse particles which could scratch it. 
If minerals that are rich in iron are abundant, 30 |Lim is not thin enough to 
enable observation and it is necessary to reduce the section to 
approximately 20 |Lim to make it sufficiently transparent. 

For scanning electron microscopy or EDX probes, the final polishing 
should be done with a 0.25 |Lim diamond paste. For high resolution 



Microscopic Analysis 261 

transmission electron microscope observations, the glass support should 
be removed and the thickness of the micro-zones (diameter 3 mm) 
reduced, these should be separated and cut with a scalpel. An argon gun 
is used for thinning. For observation of minerals oriented in the same 
plane as the slide, a microtome can also be used for cutting. Micro- 
diffraction will provide some information, but interpretation is often 
difficult on very thin samples. 

Separation on slides of micro-particles of about 50-100 |Lim is possible 
with an ultrasound probe equipped with a carbon needle on a micro- 
manipulator; the slide is observed on a reversed optical microscope. 
Micro-particles are placed on a silicon plate and are analysed by XRD 
with slow scanning. The resulting spectra are compatible with angular 
spaces and standard intensities. 

8.2.3 Grids and Replicas for Transmission Electron 
Microscopy 



Principle 

The samples can only be observed in transmission electron microscopy if 
they are less than 1 |Lim thick and if they are assembled on very thin 
conducting films. The object subjected to electron bombardment must be 
crossed by the electron beam. The acceptable thickness depends on the 
energy of this beam i.e. approximately 0.2 |Lim penetration at 50 kV, and 
3-4 times higher at 100 kV. 

The beam-matter interaction heats the sample-target which can cause 
volatilization of the pore water; the morphology of halloysites is then 
seriously damaged and there will be contamination of the gun of the 
microscope. With elements of higher atomic number, heating can be very 
intense (fusion, phase shift, sublimation, destruction of supporting film). 

The supporting film should be transparent to the electrons, sufficiently 
solid to support the sample, resistant to heat and to electrostatic charges; 
it should be no thicker than about 100 A. 



Reagents 

- Polyvinyl formal (FORMVAR, n D 20 : 1.50) 

- dichloroethane, C1-CH 2 -CH 2 -C1 

- 0.15% FORMVAR in dichloroethane solution; 

- COLLODION (nitrate of cellulose or pyroxylene Ci 2 Hi 6 N 4 0i 8 ) 

- amyl acetate (CH3-C0 2 -C 5 H n ) or butyl acetate (CH 3 C0 2 (CH 2 ) 3 CH 3 ) 

- 1% collodion solution in amyl acetate 

- 1% fluorhydric acid in water. 



262 



Mineralogical Analysis 



Equipment 



Fig. 8.3. Grid of transmission 
electron microscopy § 3 
mm (322 meshes) 
numbers: location of 
x- coordinate 
letters: location of 
y- ordinate 




- Lab glassware 

- Cu (or Ni) grids 3.05 mm in diameter (Fig. 8.3); these are also available 
covered with an FORMVAR-carbon film or with a perforated 
FORMVAR-carbon-gold film enabling direct observation of micro- 
particles 

- 1 10 mm non-magnetic stainless grips with ultra-fine points 

- plastic film for replicas 0.034 mm thick 

- micro-drying oven to melt the replicas at 45 °C 

- sampling loop. 

Procedure 



Grids 

- Place the required number of 3 mm grids on a glass slide previously 
moistened with water. Place the slide in a cupel and very gradually 
submerge it; the grids should not float 

- add a drop of FORMVAR-dichloroethane solution to the surface of the 
water; the drop should form a very thin film (<100 A) without folds or 
holes which will solidify in the air after evaporation of the solvent (the 
film will be thinner if the water temperature is close to 0°C); eliminate 
the liquid slowly to bring the film into contact with the slide and the 
grids it covers; let dry then cover the grids with flash carbon which 
reinforces the film and makes it conducting; when the grids are grey, 
they are ready to use; check their quality under a binocular microscope 



Microscopic Analysis 263 

- dilute a drop of sample suspension in water to obtain an almost clear 
liquid; treat with ultrasound to separate the particles; remove one 
micro-drop and place it in the centre of the grid 1 the grid should not be 
turned over during the operation; let dry at air temperature and 
dehydrate in a critical point apparatus if necessary. 

Replicas 

Samples which change shape during desiccation are too fragile or too 
dense to transmit the electrons but can be studied using a one or two- 
stage replica technique that results in a slight decrease in resolution. 

First the sample should be rapidly subjected to directional shading with 
gold or platinum, then the surface covered with a vertically applied 
carbon film that is both conducting and resistant. 

In this case, cover the sample with a special 0.034 mm thick thermo- 
fusible film that softens at 45°C and preserves a replica of the surface of 
the mineral. Dissolve the clay sample with its cover in a diluted 
hydrofluoric acid solution. The replica remains in the hollows of the 
sample and can be subjected to gold or carbon treatment (if this has not 
already been done). Then dissolve the polystyrene film in ethylene 
chloride. Assemble the carbon replica on a grid and observe using TEM. 
All these procedures should be carried out with extreme care. 

8.2.4 Mounting the Samples for Scanning Electron 
Microscopy 



Principle 

The samples may be massive and rough. Depending on the characteristics 
of the sample vacuum chamber, discs 20 cm in diameter and 4 cm in 
thickness (8 in. wafers) can be used, but the degasification of large 
samples is only possible in the case of compact rocks with a limited 
number of fissures. In practice, it is preferable to use smaller samples. 
The surface for observation must be a clean break in the sample to enable 
the study e.g. the plan of cleavage, crystal orientations, defects in the 
crystal lattice, the presence of occluded impurities. 

Surfaces are rendered conducting with flash-carbon or by metallization 
if micro-probe analysis is not required. Otherwise, if the SEM is 



1 The grids should be handled with forceps with ultra-fine points. 0.1 ml_ micro- 
syringes of the Hamilton type. Flame-drawn hydrophobic glass tubes treated 
with PROSYL 28 can also be used. 



264 Mineralogical Analysis 

equipped with an EDX or WDX micro-probe, flat, perfectly polished 
surfaces (0.25 |Lim) are required. 

Equipment 

- Special SEM supports 

- storage boxes 

- 5 x 5 mm calibration grids with 2 |Lim squares 

- 3 mm carbon slides to mount on SEM supports 

- pencil marker for SEM (conducting ink) 

- double-face self-adhesive ribbon with low content of volatile elements. 

Products 

- Silver lacquer 

- conducting carbon lacquer 

- a set of reference minerals for quantification. 

Procedure 

Right Wrong 

Fig. 8.4. Position of the samples on SEM ^fllTl ! QL_ I I Ik 

supports 



Slide 



Small samples can be assembled on aluminium supports by sticking 
them together with silver or carbon lacquer. In certain cases, carbon 
supports can be used, or plates of 3 mm thickness stuck on aluminium 
supports. Carbon lacquer is generally preferable for EDX micro-probe 
analysis. It is essential to locate the samples precisely (e.g. by squaring, 
marking, or marking the right direction on the support); it is also 
important to avoid creating a vacuum under the sample (Fig. 8.4) because 
this causes discontinuity, and elimination of the charges can be disturbed 
resulting in scratches on the images which renders the photographs 
unusable. The lacquer should not cover the sample or fill the cracks. 
After prolonged drying to eliminate solvents, cover the samples with 



Microscopic Analysis 



265 



flash carbon using a metal sprayer with ionic bombardment, or shadow 
with a metal deposit from an evaporator. 

8.2.5 Surface Treatments (Shadowing, Flash-carbon, 
Metallization) 



Vacuum Evaporator 

Flash carbon is often used to reinforce the FORMVAR film which 
supports the samples; it also makes the film conducting. Flash carbon 
should be applied vertically and uniformly. The micro-samples are 
sometimes only slightly absorbent and are not very visible in TEM. In 
this case the sample can be covered with a directional deposit of carbon 
whose grain is not very apparent, or be metallized with platinum or gold, 
under a tangential entry. Each space protected by a relief will appear 
shadowed. Knowing the angle of incidence, it is possible to measure the 
length of the shadow and deduce the height of the corresponding relief 
(Fig. 8.5). 



Fig. 8.5. 

Vacuum 

evaporator 

(shadowing 

with flash 

carbon 

metal) 



or 



/ V 



/\ 



s 

nrmn 



Shadow 



Sputtering Metallization 



The apparatus consists of an anticathode made of gold shaped in a ring 
whose internal diameter is longer than the length of the sample support. 
At the centre, a cylindrical magnet is connected to a magnetic field which 
forms the other pole and surrounds the anticathode (Fig. 8.6). The 
electrons, which would otherwise overheat the sample, are deviated by 
the magnetic field. 



266 



Mineralogical Analysis 



Fig. 8.6. Sputtering metalliza- 
tion apparatus (Bio-Rad - 
olaron) for SEM samples. N 
magnet, 1 Au anticathode, 
2 sample support, 3 cooling 
block, filled circle neutral 
atoms, open circle positive 
ions. When the sample 
advances in the magnetic 
field, all sides are bombarded 



: Magnet axis 




n^n 




The sample support is cooled to 4°C by a Peltier thermoelectric 
system. Heating is thus reduced and it is consequently possible to treat 
organic samples. 

The treatments are carried out under 10 1 Torr vacuum for 30-180 s. 
The applied voltage can reach 3 kV. Sweeping with a dry neutral gas 
(argon) enables elimination of residual traces of water, carbon dioxide, 
oxygen and possible oil contamination from the vacuum pumps. It is 
sometimes necessary to degas porous samples for several hours. In this 
way contamination is limited, but it is nevertheless often preferable to 
dehydrate on the apparatus at critical point before continuing 
degasification in the metal sprayer and then metallization. 

Cryo-fixing is often useful for organic matter and very frangible 
samples. 

This treatment gives excellent surface conductivity and accentuation of 
the relief of the rough samples by shadowing in SEM. 



Microscopic Analysis 267 

8.3. Microscope Studies 



8.3.1 Optical Microscopy 



Description 

Optical microscopes allow observation of objects that are too small to be 
observed with the human eye or with a magnifying glass. Direct 
observations are carried out under IR to UV radiation including visible 
radiation, and can be accompanied by photography on film (black and 
white or colour) or digitalized video images. 

The magnifying power of a magnifying glass ranges from 2 to 60 times 
and of the most powerful microscopes up to 1,500 times. The object can 
be massive or very thin (a thin slide that is covered or not) to determine 
properties of soils or soil materials using absorption-transmission. 
Covering slides protects the surface from oxidation. Covered slides can 
be used for optical observation in immersion but the slides cannot 
subsequently be used for electronic microscopy. 

Briefly, an optical microscope is composed of a stand which supports a 
mechanical mount ensuring vertical displacement of an objective, and an 
eyepiece over an object slide. 

The magnifying power and the diameter of the field characterize the 
relations between the image and the object. Lightness refers to the 
luminosity of the optics used. The depth of field and the focusing range, 
as well as the limit of resolution determine the zones where the object can 
be observed under optimal conditions for a given material. A system of 
lighting allows observation by reflection or transmission. The lighting 
can be directed for massive objects (low-voltage lamps, optical fibres). 
For very thin objects, the lighting can be concentrated into a point by 
condensers with respect to different backgrounds: pale background, dark 
background, polarized light, phase contrasts, UV (slides covered or not). 

IR microscopes use optical systems with mirrors to avoid adsorption of 
IR by the materials generally used in the manufacture of lenses. 

Polarizing Microscope 

In soil sciences, polarizing microscopes are primary tools for the 
observation of crystals and the characterization of their optical properties. 
Interference microscopes or phase contrast microscopes (invisible 
transparent objects against a pale background) are rarely used. Variations 



268 



Mineralogical Analysis 



in transmission factors can reveal structures that are invisible in natural 
light and make it possible to identify phenomena of pleochroism, isotropy 
and anisotropy of structure and mineral associations (as in forms, facies, 
cleavages, macles) using cross or slightly uncrossed polarizers. These 
microscopes (Fig. 8.7) enable observation of variations in transmission 
compared to the direction of polarization of the incidental light. A 
calibrated compensator placed in front of the analyser allows observations 
to be quantified. The choice of the objective is important (magnifying 
power, immersion or not) for the quality of soil observations. 



Sample 
(thin section) 



Goniometric 
stage 




Eyepiece 



Analyser 



Lens 



I 



I 



Fig. 8.7. Diagram of a 
polarizing microscope 



Condenser 
Diaphragm 

Polarizer 
Condenser 



— — | Diaphragm 
Source 



9 



For the study of the structure and porosity of soils, the size, shape, 
associations of individual grains, and distribution of the different phases 
are determined either on extracted phases, or on thin sections (Jongerius 
et al. 1972; Bullock and Murphy, 1980). 

Procedure 



A rotating support, graduated in degrees, makes it possible to measure the 
extinction angle and to identify primary minerals that are still present as 



Microscopic Analysis 269 

well as to specify the scale and type of weathering. Interpretation requires 
considerable experience meaning only specialists in petrography are usually 
able to do it. Orthoscopic methods are time consuming but generally more 
precise than conoscopic methods (Wahlstom 1969, Hartshorne and Stuart 
1970). 

This type of analysis is qualitative, but can be quantified by counting 
the mineral particles originating from the parent rock and minerals liable 
to weathering in the fractions previously separated by fractionation using 
particle size, density, hardness, magnetic properties, etc. The shape of the 
particles (rounded edges, sphericization of softer minerals), surface 
appearance (such as flatness of the particles, cleavages, cracks, different 
coverings) are indicators of the form of deterioration, erosion and 
transport (chemical weathering, micro-corrosion, waterice- or wind- 
erosion). 

The nature of minerals can be deduced from their colour, opacity, and 
especially from their refractive index and observable modifications in 
polarized light (e.g. pleochroism). Certain minerals have a more or less 
clear birefringence. The extinction angle can be reached with varying 
degrees of rapidity, and may be partial or complete. The shape of certain 
crystals is characteristic. 

These observations can be supplemented by scanning electronic 
microscopy after rapid mounting of the materials on double face sticking 
supports made conducting with flash-carbon (cf. Sect. 8.2.5). 

Using thin sections allows observation without disturbance of the 
in situ arrangement of the sample, orientations and associations of 
minerals, discontinuity of the mineralogical composition of a profile 
(decrease in or disappearance of certain minerals, ratio of minerals 
resistant to weathering:minerals liable to weathering giving index of 
deterioration). 

The development of concretions, nodules, the appearance of 
cementing, the presence of organic matter at different stages of 
decomposition can be observed and quantified by subsequent 
measurements using SEM combined with EDX (certain artifacts of 
preparation, like the filling of neocracks, or holes made by polishing by 
alumina are easily revealed). 

The units of organization (skeleton, plasma, vacuums) can be studied 
in detail at different scales using extracted fractions or/and thin sections: 
the skeletal components correspond to particles that are not reorganized, 
plasma corresponds to fine elements that can move and reorganize (such 
as clays and oxides), vacuums are related to porosity (circulation of air 
and solutions in soil). For the specific study of pore spaces, fluorescent 
colours can be mixed with the impregnation resin during the preparation 
of thin sections (cf. Sect. 8.2.2). 



270 Mineralogical Analysis 

8.3.2 Electron Microscopy, General Information 

All electron microscopes are based on the interaction of electrons with 
matter. The energy of an electron accelerated by a voltage V is equal to E 
= m v 2 /2 = e V (with m, v, e = mass, speed and charge of the electron, 
respectively). High energy radiation (fast electrons) can affect the level of 
the deep electronic layers of the atoms. Weak energy radiation (slow 
electrons) only affects the external electronic layers which reflect the 
chemical state of the atoms. The total effect of the electron beam is 
related to the electronic cloud of the Z electrons (e~) of the electronic 
orbitals around the nucleus of atoms. 

The following factors should be taken into account when considering 
how to change the way radiation affects matter: 

- intensity: transmitted or reflected intensity is lower than incidental 
intensity, absorption occurs 

- direction: there is scattering with loss of energy (inelastic scattering 
modifying internal structure), or without loss of energy (coherent 
elastic scattering allowing diffraction) 

- energy: as some energy is lost, reflected, transmitted or scattered energy 
is lower than initial energy. 

Losses in intensity and energy may be accompanied by modification of 
the matter due to the effect of the radiation: 

- in the case of electron microscopes with very high energy (3,000 kV), 
the sample can gradually be destroyed 

- in the case of microscopes with energy lower than 400 kV, there is 
transfer of energy by excitation of the electrons, thermal vibrations, 
particle ejection, and emission of secondary radiations usable for 
quantification. The heating effect produces phonons. Some chemical 
effects are reducing (e~ gain). Chemical bonds can be ruptured. 

When the transfer of energy is higher than the threshold of 
displacement (between 15 and 30 eV), the effects of irradiation can cause 
atomic displacements. With electronic corpuscular incidental radiation of 
sufficiently high energy, an orbital electron in the deep atomic layers can 
be ejected with a kinetic energy corresponding to the difference in the 
energy lost by the incidental radiation and the electron's own energy 
(secondary electrons). As the excited state is unstable, the atom 
subsequently returns to a fundamental state; there is release of X-photons 
or Auger electrons (relaxation phenomena). 

With electromagnetic radiation such as incidental or re-emitted X-rays 
photoelectrons are obtained (IR- to UV-photons, cathodo-luminescence). 
Electron microscopes can be classified at two levels depending on the 
geometry of the sample: 



Microscopic Analysis 271 

- massive samples which, as they are very thick, can be analysed only by 
the signals that come from their surface by reflection 

- samples with a critical thickness that allows radiation to cross them 
(micro-crystals, thin films, etc.), in which case measurements can be 
made by transmission. 

However, progress in instrumentation has led to changes in this 
dichotomy with the appearance of hybrid apparatuses allowing 
measurements on thin samples that use both processes. The following 
types of equipment are available: 
-traditional transmission electron microscopes TEM (possibly with 

additional functions in transmission scanning mode) 

- scanning transmission electron microscopes (STEM) 
-microscopes with scanning by reflection (conventional scanning 

electronic microscopes: SEM) 
-microscopes with scanning by reflection with differential vacuum 

where the observation chamber is under partial vacuum (environmental 

scanning electronic microscopes: ESEM). 

Each type of apparatus allows complementary measurements. The 
apparatuses are suitable for either very high resolution, or, with multiple 
configurations, for a range of different chemical and physico-chemical 
approaches. The signals obtained are complementary in the energy fields. 

8.3.3 Transmission Electron Microscopy, Micro-diffraction 



Principle 

Transmission electron microscopes use an incidental electron beam 
which, while crossing a very thin sample, provides information on the 
shape and structural distribution of elementary soil particles. The 
interaction of the electron beam with the matter results in images and 
micro-diffraction spectra (and enables selection of elementary chemical 
analyses as complements to the different electronic micro-probes in 
STEM). 



272 



Mineralogical Analysis 



a Lb 



Source 

Photons Electrons 

Condenser 

(Lens) (Coil) 



(Thin Object 
section) 



(Lens) 



Lens 



= ^AIignment 
— I coils 



Astigmatism 
correction 



Atmospheric 
pressure 



Limit of 
resolution 



Fig. 8.8. 

Comparison 
of an optical 
microscope 
and a trans- 
mission 
electron 
microscope 



2 000 A 2^ 

1^ — - Intermediary image 

— ^- Eyepiece 



Lens of 
chamber 



A , 



B" 



Final image 




Projector | 
coil 



The geometry of an electron microscope can be compared to that of an 
optical microscope (Fig. 8.8). A source of electrons (high voltage electron 
gun) replaces the source of photons. A system of illumination with 
electromagnetic condensers concentrates the electron beam on the object; 
an electronic objective forms an intermediary image which is captured by 
projection lenses to form the final image on a fluorescent screen or a 
photographic device. 

Not all the radiations generated by the incidental electronic beam 
(Fig. 8.9) are used, since the apparatuses generally have only 1-2 
sensors. 2 X-photonic radiation (1, Fig. 8.9) can be collected by an EDX) 
detector (with Si-Li detection, or diodes without windows for analysis of 
light elements such as nitrogen and carbon), or WDX (with crystal 
monochromator). 



At their maximum configuration, some top-of-the-range commercial analyzers 
include up to five sensors. 



Microscopic Analysis 



273 



IR-UV-visible photonic radiation (2, Fig. 8.9) can be detected by 
cathodoluminescence. The scattered electrons (5) can be analysed by 
electron energy loss spectrometry (EELS) and enables analysis of light 
elements in STEM. Back-scattered electrons (6), secondary electrons (7), 
and Auger electrons are used for SEM images and transmitted electrons 
( 1 2) for TEM and STEM. 



1 : X photons 



2: IR, visible, UV 
photons 

3: Sample 
current 



Incident electron beam 




4: Transmitted X-rays 

5: Inelastic scattering 
of electrons 
(energy loss) 



6: Back-scattered electrons 
7: Secondary electrons 

8: Auger electrons 



9: Phonons, ultrasounds 



10: Ions, desorbed atoms 
(simulated desorption) 

11 : Elastic scattering of electrons 
bremsstrahlung 



12: Transmitted electrons 

TEM-STEM (observations against 
a light background) 



Fig. 8.9. Types of radiations emitted during the bombardment of a sample by an 
electron beam 



The choice of a transmission electron microscope depends on the 
nature of the observations required (magnifying power, high resolution, 
the need for high voltage for excitation or penetration, possible chemical 
quantification) and the cost, but also the versatility and the possibility to 
up-grade the equipment, its ability to cover the whole range of 
magnifying power including weak magnifying power, the degree of 
automation and ease of use, the quality and the cleanliness of the vacuum, 
contrast and performance against a dark background, possible coupling 
with systems for chemical analyses and image analysers, etc. The annual 
cost of maintenance contracts and consumables should also be taken into 
consideration, as these can represent 3-6% of the initial purchase price. 




274 Mineralogical Analysis 

Emission of electrons 

An electron gun is the source of electronic radiation. Generally, radiation 
is caused by the thermoelectronic emission of a filament of tungsten 
heated to 2,500°C (or of a tip of lanthanum hexaboride heated to 
1,600°C) which forms the cathode (Fig. 8.10). 

Fig. 8.10. Thermal-emission electron 
gun by: (1) tungsten (on the left 
or LaB 6 (on the right) filament, (2) 
focusing electrode polarized negati- 
vely with respect to the filament 
(Wehnelt), (3) anode, (4) cross- 
over (10 to 50 |im). Wehnelt is 
carried to a negative potential of a 
few volts to push the emitted 
electrons out of the axis and to 
concentrate them in a narrow beam 

The tungsten filament has a diameter of approximately 0.1 mm, is V 
shaped and pointed at the end to focus the emission. The filament is heated 
to a high temperature under vacuum and is subjected to a work voltage of 
4.5 eV. The conduction electrons can then cross the barrier of potential and 
an electronic cloud is formed. These electrons are accelerated by a potential 
Vq. An electron beam is obtained whose energy is E = eV . The emitted 
electrons move into the column at constant speed thanks to the high electric 
potential between the filament and the anode (supply voltage of the anode). 

A cathode made of lanthanum hexaboride (LaB 6 ) with a work voltage of 
2.7 eV, i.e. weaker than that of tungsten, can be used at a lower temperature 
(approximately 1,600°C); brightness is then considerably improved. 
However, the vacuum must be changed to 10 -7 Torr, and the reactivity of 
LaB 6 with certain metals can be awkward. Electron guns with field 
emission are also available whose brightness is much greater than that of 
thermoelectronic guns and whose energy dispersion is reduced. 

The incidental beam of electrons emitted by the electron gun (<1 mm of 
the cross-over) crosses the column of the microscope following the optical 
axis. Electromagnetic lenses are solenoid and consequently generate a 
magnetic field that focuses the electrons. An external shield prevents the 
dispersion of this magnetic field. The usual acceleration voltage varies from 
50 to 1,250 kV, but can reach 3,000 kV. In practice, microscopes are 
available with (1) voltage of less than 100 kV, (2) medium voltage of 
between 200 and 500 kV, (3) high and very high voltage electronic 
microscopes (HVEM), 1,250 kV and above. Those in group (3) are very 
expensive, very voluminous and require special safety equipment. 



Microscopic Analysis 275 

Observations 

In TEM mode, high resolution electronic microscopes (HREM-HRTEM) 
(200-300 kV) can be equipped with 15 A probes which enable the study 
of the morphology of the samples at different scales, direct observation of 
the atomic structure of a crystal and of the stacking of atoms (1.5 A at 
400 kV) on micro-samples where XRD is not efficient. These 
microscopes are thus useful to study problems of fundamental 
crystallography, phenomena of deterioration (germination and crystal 
growths, transformation of the phase that is amorphous to X-ray into 
crypto-crystalline and crystalline phases in the repetitive structures of 
clays). For example, interstratifications of mica-chlorite and minerals of 
7-14 A were studied by Amouric (1987, 1990) and Amouric et al. 
(1988), mica-kaolinite associations by Ahn and Peacor (1987). 3 

Using these techniques, it is possible to detect the planar defects, relic 
layers, and pale fringes of the interlay er levels. Care should be taken with 
high resolution to ensure that the high electron energies do not cause 
irradiation damage due to powerful vibrations of electron matter. 

Micro-diffraction 

In mineralogy, micro-diffraction of electrons is generally carried out 
simultaneously with the observation of images of normal incidence. The 
objects are prepared on micro-grids at sufficiently low density to insulate 
the elementary particles in the same way as for imagery. 

Micro-diffraction can be performed with the majority of the TEMs 
simply by adjusting the diaphragm. The fast electron beam at low 
wavelength and high energy (20-60 keV or more) strikes the extremely thin 
micro-crystal (<100 A); when the angle of incidence on the reticular levels 
is in agreement with Bragg 's law (cf. Chap. 4) spots of diffraction are 
observed (Fig. 8.11). On submicro-samples, the spectra obtained are 
characteristic of single-crystal structures. Such a detailed view of crystal 
arrangements and defects cannot be obtained with traditional XRD 
(cf. Chap. 4). 



3 For these very fine studies, the zones of interest are selected on thin slides with 
SEM at magnifying powers of about 10,000-20,000. These zones are separated 
on sections thinned with an ultra-microtome and an argon gun to ensure they 
are sufficiently transparent for the electrons in HRTEM. 



276 



Mineralogical Analysis 



Selected 
| zone 

I dS I 



n w 

! / : I • : / 



-A 



Focal 
plane 






d'S 



/! 



j20 I 



Intensity 




Beam of 

electrons 

Micro-diffraction 




Density of rings 



Fig. 8.11. Micro-diffraction by adjustment of the diaphragm 

Micro-diffraction by adjustment of the diaphragm uses a diaphragm 
placed in the image plane which defines a reduced active surface of 
approximately 1 |Lim 2 . This method enables readable spectra of oriented 
micro-crystals to be obtained, but the very small wavelength of the 
electrons induces weak angles of diffraction and associated intensities 
that are different from the methods of traditional X-ray diffraction 
described in Chap. 4. 

As the sample is very thin, the diffraction spots obtained are 
characteristic of single-crystal structures. This method is often used as a 
complement to traditional XRD. On polycrystalline materials like clays, 
annular spectra can be obtained in about a minute. 

Special TEM Techniques 



Visualization of Charges with Colloidal Gold 

Principle 

As colloidal gold is opaque to electrons, it is used as a tracer to reveal edge 

charges or structural defects in crystalline structures (Photo 8.1 left). 

Equipment and reagents 

- TEM grids 3.05 mm in diameter covered with an FORMVAR film and 
carbon 

- 5 nm particle size colloidal gold solution (store in the refrigerator 
protected from light). 



Microscopic Analysis 



277 




Photo 8.1. Special transmission electron microscopy techniques. On the left, 
highlighting of edge charges in kaolinite by colloidal gold (see 
procedure in text of this section), on the right view in paraglyph 
(see procedure in below), photographs (x 90,000), Gautheyrou J., 
IRD mineralogical reference set, Bondy, France, unpublished data 



- Suspend the colloidal gold solution by agitation 

- take 0.5 mL of gold suspension and mix in a glass tube with 0.5 mL of 
sample of low density in order to obtain well separated minerals; leave 
in contact for a few minutes; agitate and put a micro-drop on a 3 mm 
grid., 

Allow to dry in the air and view under a TEM with a magnifying 
power of about 60,000. Gold preferentially migrates towards the rupture 
or crystallization zones and reveals the modes of assembly and the active 
sites of certain clays. 

Development in Paraglyph 

Principle 

The aim is to obtain a pseudo relief by superposition, with a tiny shift 

of negative and positive transparencies of the same image (Photo 8.1 

right). 



278 Mineralogical Analysis 

Equipment and products 

- Negative film with strong contrast, format at least 6.5 x 9 cm 

- transparent positive film 

- photographic development products (developer - fixer). 

Procedure 

- Choose a clear negative of the image 

- by contact trace the image on a positive transparency of similar density 

- superimpose the negative and positive and find the optimal shift needed 
to obtain an effect of relief 

- draw by tracing or by enlargement. This type of image makes it 
possible to see coverings of particles more clearly and the effect of 
relief can be spectacular. 

Opacification of Samples that are "Transparent" to Electrons 
Minerals rich in iron are very opaque to electrons and can cause problems 
if they are too thick as the resulting images are very strongly contrasted 
and no details are visible. 

On the other hand, certain very fine minerals like allophanes are 
practically transparent to electrons if they are present in low concentrations. 
These preparations can be opacified with a lead salt (PbCl 2 at 1% in water). 

The sample is left in contact with lead solution for one hour, then 
washed, suspended again and diluted to prepare a TEM grid. 

Scanning Transmission Electron Microscopy 

The electron gun and the condenser system used to produce the electron 
beam are based on a principle that is similar to traditional TEM, but in 
TEM the signal is transmitted to the image plane observable on a 
fluorescent screen via a system of electronic lenses, whereas in scanning 
transmission electron microscopes (STEM), the signal is directly 
collected by electron or X-ray detectors, and transmitted on-screen 
(Fig. 8.12). 

In true STEM, an electron gun with field emission, whose cross-over is 
about 5 nm and whose brightness is more than 1,000 times higher than 
that of a traditional tungsten source, provides an electron beam which 
crosses a condenser giving a reduced image of the source (micro-probe). 



Microscopic Analysis 



279 



Fig. 8.12. Diagram 
of a scanning 
transmission 
electron 
microscope 



Scanning 


t 


generator 




i 


□ 










Screen 








Energy 
analyzer 




Amplifier 







Field emission 
electron gun 

Condenser 



Sample 



Electron 
detector 



This probe scans the surface of the sample by means of a deflecting 
coil. The electrons transmitted or diffracted by the sample are collected 
on a detector with a response that is proportional to their intensity. After 
amplification, an image is created on the screen stage-by-stage in 
synchronization with the scanning generator. 

Electron guns with field emission are very sensitive to contamination. 
They require an ultrahigh "dry" vacuum (10~ 10 Torr), which proscribes 
the use of oil diffusion pumps for the secondary vacuum. In spite of the 
use of cryoscopic traps, the gun can still break down because of traces of 
oil. 

Very high spatial resolution can be achieved. This equipment can also 
be equipped with energy analysers such as electron energy loss 
spectrometers (EELS). They can carry out analyses on surfaces of the order 
of one nanometer on all the elements of the periodic table ( 3 Li to 92 U) on 
submicroscopic samples. 

Dedicated STEM are not the most widely used, many manufacturers 
prefer to sell hybrid TEM equipped with complementary STEM which 
perform excellently for a price that is 2 at 3 times lower. 



8.3.4 Scanning Electron Microscopy 



Scanning Microscopes by Reflection, Microprobes 

The concept of the scanning electron microscope (SEM) and that of 
electronic micro-probes (EM) are complementary, EM comprising probes 
of less than 1 |Lim optimized for X-ray analysis. 



280 Mineralogical Analysis 

The thermionic electron gun is subjected to a negative voltage of 10- 
50 kV. The sample is placed on a goniometric precision support (a 
binocular magnifying glass enables visual location of the point of impact 
on the microprobe). 

Electromagnetic condensers form the image of the probe which is 
projected on the sample. The probe is moved by deflection of the beam. 
A massive sample can be 2-4 cm thick and have a diameter of 20 cm 
(8 inches wafer). A large-capacity sample chamber requires a clean vacuum 
system with a strong flow (turbomolecular pump). The magnifying power 
is the ratio of the amplitude of the scanning of the image (fixed) to the 
amplitude of the scanning of the object (variable). 

Electron-matter interactions (secondary and back-scattered electrons, 
X-ray, Auger electrons, photoluminescence, transmitted electrons) can be 
used for analytical measurements. 

The image is created stage-by-stage (pixel by pixel) and allows 
digitalization and treatments using an associated data processing system. 
The creation of the images is based on two modes: 

- in secondary electrons mode the incidental primary radiation of the 
electrons loses energy in contact with the matter; part of the energy is 
restored in the form of secondary electrons which cross the grid of the 
collector and are then accelerated in the field of the scintillator; an 
exploitable signal is obtained which is mixed with the back-scattered 
electrons which are able to cross the diaphragm of the detector 

- in back-scattered electrons mode the electrons are collected by the 
collector of a scintillation detector; the signal is rather weak but 
detection is improved by using a semiconductor detector in the shape of 
disc that is perforated in the centre which is placed above the sample; a 
device installed in two or four different sectors makes it possible to 
create a topographic contrast. 

The chemical composition of the sample sometimes varies in a random 
way because the rate of penetration is very low. The shade of grey is 
related to the atomic numbers of the elements observed. 

The resolution is about 20-100 A depending on the element observed. 
The intensity of the electron beam and of the scanning conditions is 
chosen to ensure maximum resolution and an optimal signal-to-noise 
ratio for a given magnifying power. Even in the best conditions strong 
incidental energy (approximately 30 keV) prevents very fine details from 
being observed, but generally reasonably good results are obtained. On 
the other hand, if the material is slightly conducting and cannot be 
sprayed with metal, it may be better to reduce the charge by using energy 
below 5 keV. 



Microscopic Analysis 281 

The diaphragm should be selected to obtain a suitable depth of field, as 
well as to allow adjustment of the work distance if the relief of the 
sample is significant. 

Environmental Scanning Electron Microscopy 

These microscopes enable high resolution images to be obtained by 
reflection on samples preserved in their natural moisture, without 
degassing or surface conducting treatment. Some environmental 
investigations can be made without deformation or transformation of the 
sample. Two systems are used: 

- low vacuum scanning electron microscopes (LV-SEM) are relatively 
simple and can be used in conventional SEM; they enable a partial 
vacuum of about 2-4 Torr to be created in the sample chamber; they are 
generally equipped with a detector of back-scattered electrons 

- environmental scanning electron microscopes (ESEM) are dedicated 
microscopes which enable a high vacuum to be created in the electron 
gun (10~ 7 Torr) and simultaneously a reduced vacuum of near 
atmospheric pressure to be created in the observation chamber. This 
difference in vacuum is obtained in stages with progressive reduction in 
pressure. 

The distance between the sample and the output of the electron beam 
under high vacuum must be as small as possible in order to avoid a 
reduction in performance. In conventional SEM, more than 95% of the 
electrons do not undergo dispersion. In environmental SEM with the 
sample at a short distance from the beam output under a pressure of 1 
Torr, the proportion of non-dispersed electrons can reach 90%, but 
decreases with the number of gas molecules in the trajectory of the beam. 

A specific gaseous secondary electron detector (GSED) enables the 
quality of the image to be improved by discriminating the back- scattered 
electrons and the secondary electrons resulting from the interactions of 
the electrons of the beam and the atoms of the sample. There is no artefact 
of charge as in conventional SEM (e.g. ionization of gas, production of free 
electrons, or creation of positive ions compensating for the negative 
charges). This detector is not sensitive to light or to temperature. 

The atmosphere in the sample chamber can be controlled at the same 
time as the pressure and the temperature and enables observations in a 
gaseous medium of almost constant composition. Interpretation of the 
images requires adaptation to phenomena such as condensation on the 
minerals (for example rounding of the angles), the presence of interstitial 



282 Mineralogical Analysis 

water or pollutants and the determination of gas balances. Quantitative 
measurements by EDX (cf. Sect. 8.3.5) are possible. Many applications in 
soil science, especially in studies of organic matter, clayey materials, and 
micro-organisms are now possible using ESEM (Mathieu 1998, Leroux 
and Morin 1999), for example: 

- physical problems involved with expansible minerals, allophane soils 
with high water retention, structure, texture, porosity, aggregates, transfers 
between the soil and the environment, dehydration and hydration 
processes, soil shrinkage, compression, adhesiveness 

- effect of heat or chemical treatments, fusion, sublimation, growth of 
crystals, stabilization of structure, tests under constraint 

- dynamics of the degradation of organic matter, micro-fauna. 



8.3.5 Ultimate Micro-analysis by X-Ray Spectrometry 



Energy Dispersive X-Ray Spectrometry 

Micro-determinations are usually carried out by X-ray fluorescence 
spectrometry (cf. Sect. 3 1 .3.2, Chap. 3 1) by means of an EDX spectrometer. 
This system (Fig. 8.13) enables plotting of charts of elementary 
qualitative distribution at the surface layer and on approximately 1 |Lim 
thickness. It is better to use almost flat surfaces; for accurate quantitative 
analysis, surfaces have to be polished to 0.25 |Lim to limit possible 
topographical effects. 

A fixed probe can be used if less precise quantitative micro-analyses is 
needed than that obtained with dedicated analytical probes, but ZAF 
matrix-correction software (Z: atomic number, A: absorption, F: 
fluorescence) enable improvement of the results. These analyses can only 
be performed on elements heavier than n Na. Light elements require the 
emission of Auger electrons; but the ultra-high vacuum of 10~ 10 Torr 
required in Auger spectroscopy cannot be obtained with normal scanning 
microscopes. A SAM 4 microscope is required where the vacuum is 
obtained with an ionic pump. 

Wavelength Dispersive X-Ray Spectrometry 

The source of X-rays emitted at the electron beam-matter interface is 
placed on a focusing circle called "Rowland circle" (Fig. 8.13). Detection 



4 SAM = Scanning Auger Microscope. 



Microscopic Analysis 



283 



is carried out by moving the crystal analyser and the entry slit of the 
detector (counter with proportional action) along the circle. The detector 
must be at the effective focal spot (2 # compared to the incidental beam). 



Liquid 

nitrogen 

cryostat 



Incident beam 

of electrons E = 100 - 3,000 kV 

Electrons 



Crystal 



Measurement 
electronic 




Preamplifier 



SS— U detector 
EDS 



WDS Detector with 

proportional 
action 



Fig. 8.13. Microprobes with dispersion of energy and wavelength: EDS, EDX: 
energy dispersive X-ray spectrometry, WDS, WDX: wavelength 
dispersive X-ray spectrometry (Rowland circle, effect of the defocusing 
of the electron beam, - Bragg angle, A# = deviation of the Bragg 
angle caused by defocusing) 

To carry out quantitative analysis, the direction of measurement and 
the opening must be constant. In practice, the angle of reflection cannot 
exceed the 5-70° range. It may thus be necessary to use four crystal 
analysers (Microspec-USA system) at different reticular distances 
(Bragg 's law) to cover the range of wavelengths accessible with this 
approach (lithium fluoride, LiF, Pentaerythritol, PET, rubidium acid 
phthalate, RAP, lead stearate, STE). The WDX system is tending to be 
replaced by the faster EDX system. 

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Amouric M (1987) Growth and deformation defects in phylllosilicates as seen 
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Amouric M (1990) Etude de l'interstratification mica-chlorite par microscopie 
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Amouric M, Bianetto T and Proust D (1988) 7.1 and 14 A mixed layer 

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Wright D, Stanley D, Chen HC, Shultz AW and Fang JM (1990) A frame based 

expert system to identify minerals in thin section. Microcomputer 

applications in geology II. Pergamon Oxford, 289-299 
Zhurov AV (1990) Preparation of polished sections for the study of soil pores 

and their differentiation by size. Pochvovedenize, 8, 144-147 



286 Mineralogical Analysis 

Sludzian G and Galle P (1992) Cartographie de materiaux et d'echantillons 

biologiques par microscopie ionique a balayage. La vie des sciences, 

Compte rendu serie generate, 9, 157-177 
Gribble CD and Hall AJ (1993) Optical mineralogy : principles and practice., 

Chapman and Hall London 
Fitzpatrick EA (1993) Soil microscopy and micromorphology., Wiley 
Ringrose-Voase AJ and Humphreys GS (1994) Soil micromorphology, studies in 

management and genesis ; Proceedings of the IX international working 

on soil micromorphology., Elevier Science Ltd 
Lavoie DM, Little BJ, Ray RI, Bennett RH, Lambert MW, Asper V and 

Baerwald (1994) Environmental scanning electron microscopy of 

marine aggregates. J. Microscopy, 178, 101-106 
Vempati RL, Hess TR and Cocke DL (1996) X-ray photoelectron spectroscopy. 

In Methods of soil analysis, Sparks DL ed., SSAA book series No 5 
Jonhson RA (1996) Environmental Scanning electron microscopy - An 

introduction of ESEM, Philips Electron Opt., -F EI, 55p 
Mathieu C (1996) Principle and application of the variable pressure SEM, 

Microscopy and analysis, 43, 13 
Pichler H, Schmitt-Riegraf C and Hoke L (1997) Rock-forming minerals in thin 

section., Chapman, London 
Hitachi, (2000) Low temperature microscopy using a cooling stage. Hitachi 

technical data, 62 
Astley OM, Chanliaud E, Donald AM and Gidley MJ (2001) Structure of 

Acetobacter cellulose composites in the hydrated state. Int. J. Biol. 

Macromole, 29, 193-202 
Slowko W (2001) Secondary electron detector with a micro-porous plate for 

environmental SEM, Vacuum, 63, 457-461 
Tai SSW and Tang XM (2001) Manipulating biological samples for 

environmental scanning electron microscopy observation. Scanning, 

23, 267-272 



Part 2 



Organic 
Analysis 



Physical Fractionation of Organic Matter 



9.1 Principle and Limitations 

9.1.1 Forms of Organic Matter in Soil 

Many different organic fragments can be distinguished in soils, most of 
which are of plant origin (living or dead roots, fragments of wood fibre, 
fragments of stems and dead leaves) and others of animal origin (e.g. 
cadavers, faecal pellets, earthworm casts) from macro-fauna such as 
insects, arachnida, myriapodes, Crustacea, gasteropods or earthworms. 

Increasing the scale of observation, smaller organic fragments e.g. 
filamentous roots, partially decomposed animal debris, nematodes, fungi, 
and algae can be identified with a magnifying glass. 

The observation of other micro-organisms (e.g. bacteria, actinomyces, 
protozoa) and debris of animal or plant origin that are increasingly 
incorporated in organomineral colloids requires a higher power of 
magnification. 

Initially soil organic matter (SOM) thus appears to be a continuum of 
increasingly fine fragments that can be physical fractionated. 

9.1.2 Principle 

The methods of fractionation described in this chapter include both manual 
or mechanical sorting, and the use of physical techniques for density 
fractionation, particle-size fractionation by sieving, and analysis of 
sedimentation. The methods used resemble those used in fractionation of 
mineral particles (cf Chap. 2). 



290 Organic Analysis 

Manual sorting is used especially for studies on live roots in the soils. 
Sorting is facilitated by floating using - for example - water elutriators 
(cf Sect. 9.2.2). 

Density fractionation is based on the difference in density between 
matter of plant origin (close to 1) and of mineral origin (around 2.65 for 
primary minerals). Theoretically, it is thus an ideal technique for the 
separation of fragments of plant origin that are not decomposed in the soil. 
This type of measurement is now widely used in studies on the dynamics 
of carbon in soils. Some compartment models were established with data 
obtained by densimetric separation (Pansu and Sidi 1987, Arrouays 
1994). These techniques are described in Sect. 9.2.4. However, 
depending on the type of soil, the density fractionation method may be 
hindered by the close associations between mineral and organic particles. 
The technique can be improved by combining it with particle size 
fractionation (Sallih and Pansu, 1993) and with a range of dispersion 
techniques described in Sect. 9.2.3 of this chapter. 

The aim of particle-size fractionation is complete separation of the 
organic components of the soil. Ideally, coarse fractions of more than 50 
|um would contain intact plant debris, silt fractions of 50-2 |um (or 
20-2 |um) would contain cells and microbial fragments, coarse clays of 
2-0.2 |um would contain organic matter of the organomineral complex, 
and finally fine clays of 0-0.2 |um would contain recently formed 
metabolites. Some approaches, such as dating (Anderson and Paul, 1984) 
or isotopic 14 C measurements (Hassink and Dalenberg 1996) or 8 13 C 
(Puget et al. 1995) appeared to partially confirm this theory. Other studies 
showed that micro-organisms and organic materials are closely associated 
with mineral colloids, and consequently "clean" fractionation of the 
biological components of soils is impossible (Ahmed and Oades, 1984). 
Certain review studies (e.g. Christensen 1992, Feller 1994) did, however, 
identify three main classes of organic matter: 

- a plant debris compartment (>20 |Lim) that is not closely associated with 
mineral sands with a relatively high C:N ratio (15-25) or with a high 
xylose to mannose ratio, indicating that this organic matter is of plant 
origin 

- an organic silt complex including a mixture of soil organic matter 
(SOM) of plant and fungal origin, mineral silts and very stable organo- 
mineral micro-aggregates; C:N and xylose:mannose ratios are lower 
than in the previous compartment; the origin of this organic matter is 
not as clear as that of coarser matter 



Physical Fractionation of Organic Matter 291 

- an organic-clay compartment (<2 |Lim) rich in amorphous SOM; this 
compartment is humified and closely associated with the mineral 
particles; C:N (8-11) and xylose :mannose ratios are lower, suggesting 
that the origin of this organic matter is probably microbial. 

9.1.3 Difficulties 

Particle-size fractionation uses sieving techniques (generally wet sieving) 
to separate particles until 50 or 20 |Lim. The separation of the finest 
particles requires sedimentation techniques similar to those described in 
Chap. 2 for mineral fractionation. However, an additional difficulty to 
take into consideration is particle density with respect to the Stokes law 
of sedimentation (cf Chap. 2). The average density of 2.65 used for 
mineral particles is not appropriate for organic fragments (Elliott and 
Cambardella 1991). However, at this particle-size, organic matter is often 
closely associated with mineral particles. As SOM content is relatively 
low compared to mineral particle content, one can consider that the 
densities of the mineral fractions are not significantly modified. 

The main difficulty in physical fractionation by density or particle size 
separation lies in the close association between minerals and organic 
matter resulting in different types of aggregates (Fig. 9.1). These 
aggregates have to be broken down and the organic components released 
without destroying them. Sect. 9.2.2 of this chapter discusses various 
techniques of dispersion at some length, and describes their limits and 
comparative interest. 

Preparation and especially rewetting of the sample involves a risk of 
modifying the organic constituents. The samples are generally dried 
before storage at the laboratory. Drying, together with other preparation 
techniques (Pansu et al. 2001), stops the organic functioning of the soil 
resulting in deactivation or death of micro-organisms. The treatment is 
brutal and it is preferable to slow down microbial activity by cold storage 
or even better by freezing the fresh soil. 

Whatever the technique of conservation used, stopping the biological 
activity is necessary to preserve the soil organic state at sampling, as the 
kinetics of evolution of certain organic components are higher than that 
of the main inorganic components. 

However, rewetting the soil starts biological activity again. Rapid 
growth of the populations of micro-organisms supplied with the plant 
debris which are released from their clay protection during the 
preparation of the samples (effect of grinding) and become available for 
micro-organisms, as well as by consumption of the microbial biomass 



292 



Organic Analysis 



killed during these operations. Part of this carbonaceous source is then 
mineralized or transformed. 

There is thus a risk of changing the organic contents of the soil by 
rewetting of the samples which is required by most of the physical 
fractionation techniques described later. However, this risk is limited in 
the presence of a great excess of water, since most active food chains are 
essentially aerobic. The risk can be also limited by the use of reagents 
that are unfavourable to the growth of micro-organisms and by 
performing fractionation as rapidly as possible after moistening. 



Structural aggregate 



Sand 



Bacterial cells . 



Root debris 



Organo- 
mineral 
complex 
bonds 
organic matter- 
cations-clays 




Polysaccharides 



Microaggregate 
50 urn 



Fig. 9.1. Formation of organomineral complexes, micro-aggregates and structural 
aggregates (after Bruckert 1994) 



Physical Fractionation of Organic Matter 293 

9.2 Methods 

9.2.1 Classification 

Methods for physical fractionation of organic components can be 
classified in three main groups: 

- separation of the plant roots; 

- separation by density; 

- fractionation in particle-size ranges. 

When using these methods, the structure of the soil material should be 
kept in mind i.e. humified fine organic matter associated with inorganic 
matter forming organomineral complexes. These complexes involve 
bonds between solid particles resulting in the formation of different types 
of aggregates in the soil. Some of the components that have to be 
separated are imprisoned in these aggregates (Fig. 9.1). The difficulty in 
fractionation thus consists in splitting up these aggregates without 
destroying the components that have to be measured. The principal 
techniques for aggregate dispersion are described and commented in Sect. 
9.2.3. 

9.2.2 Extraction of Plant Roots 



Objective and Principle 

This type of extraction is useful to measure root production in the soil. 
Indeed the production and turnover of roots is one of the most significant 
inputs of carbon in the soil, the other inputs being root exudation and 
above-ground necromass production by the plant. The study of the carbon 
balance in the soil and in the atmosphere has been the object of intensive 
research and methodological compilations e.g. Anderson and Ingram 
(1989) that describe methods to estimate organic inputs in the soil. 

Root extractions are also useful for observations of plant physiology 
such as classification of roots, estimation of their weight and length, 
chemical analyses, biological associations with fungi and bacteria. 

Separation is generally carried out manually but several types of 
elutriation apparatuses are available. 

Procedures 

Extraction is performed on intact samples from blocks or cylinders of 
soil. The samples must be stored in polyethylene bags at low 
temperatures or even better frozen. If a freezer is not available they can 
be dried and rewetted before washing, but the best approach consists in 



294 Organic Analysis 

washing the roots immediately after returning to the laboratory from the 
field. 

In addition to organic matter content, the texture of the soil, 
compaction, and structure affect the difficulty of the extraction to a 
varying extent. The simplest method consists of gently washing the 
wetted samples with water on a sieve whose mesh size differs with the 
author: 2 mm for Abo (1984), 0.5 mm for Anderson and Ingram (1989); 

The material remaining on the sieve can be washed with water and 
separated by decantation. To remove all the fragments, the residue often 
has to be sorted manually under water in flat containers. This work may 
require a binocular magnifying glass and very fine forceps. The difficulty 
of the work also depends on the type of soil and roots. 

Many machines have been described that wash roots; most separate 
roots from soil by elutriation, i.e. washing the debris accompanied by 
their separation by flotation on a 0.5 mm sieve located far from the heavy 
particles. Fig. 9.2 shows a diagram of the apparatus designed by Smucker 
et al. (1982) which is based on the principle of hydropneumatic 
elutriation. 

The apparatus built by Bonzon and Picard (1969) is suitable for the 
separation of roots from intact soil sampled in the form of cylinders or 
monoliths. It is composed of a set of 4 double sieves made of wood with 
a brass screen with a rectangular section (50 x 60 cm) and a cylindrical 
bottom. The top sieve has a mesh of 1 . 1 8 mm, and the bottom sieve of 1 .4 
mm. The sieves are set in a wooden frame that is moved backwards and 
forwards at 12.5 oscillations min -1 by an engine with a crank-connecting 
rod system. Samples of a volume of around 2 L are placed on the top 
sieve and jet water is directed onto the sample. Slurry is evacuated over 
the top of the raised edge of the top sieve. Once washing is complete, the 
contents of the sieves are transferred to a funnel equipped with a sieve 
with a very fine mesh. 

This funnel contains the organic fragments but also stones and gravels 
with a diameter of over 1 .4 mm. If there is a lot of gravel, the organic 
fragments should be separated using a strong jet of water directed at the 
base of the funnel and transferred onto a sieve placed below. 

After mechanical fractionation of soil and roots, it may be necessary to 
manually sort the roots from the other organic debris and this operation 
can take several hours. Consequently there is no "ideal" machine that 
eliminates all manual operations. 

All separation methods result in losses of fine roots and washing water 
and residues should be checked periodically to quantify these losses. 



Physical Fractionation of Organic Matter 



295 




o 



B 



O 



b£Vi 



£Z^ 



Ao 



Water 



Air 



t 



Fig. 9.2. Diagram of an apparatus for the separation of the plant roots from soil by 
hydropneumatic elutriation (after Smucker et al. 1982). A: high energy 
water washing chamber, B: elutriation chamber, C: transfer tube, D: 
first sieve with weak kinetic energy (840 jim), E: second sieve 
(420 |xm). Tube C is separated from B for cleaning and to introduce a 
new sample. The roots are transferred from the weak energy sieve 
(D) by reversal and washing onto the fine sieve (E) 

Soaking for one night in an aqueous sodium hexametaphosphate 
solution accelerates the process of washing roots in clay soils, but the 
chemical action may discolour the roots and break down certain plant 
tissues thereby rendering subsequent identification of live roots 
impossible. This type of pretreatment can also interfere with chemical 
analysis of the roots. In addition, all these treatments can damage the 
contents of tissues and it is consequently preferable to separate a sub- 
sample of roots by hand and to wash the roots carefully with a minimum 
of water to enable accurate chemical analysis. 

The washed roots can be stored in the refrigerator in sealed 
polyethylene bags but freezing is preferable. A small quantity of 
bactericide such as thymol can also be added. 



296 Organic Analysis 

Dry matter weight and organic carbon and nitrogen content (cf. Chap. 
10) can be measured after drying at 70°C for 48 h. Bonzon and Picard 
(1969) also measured the specific surface of the roots in addition to dry 
weight. Progressive calcination up to 550°C with successive temperature 
steps enables determination of the ash weight of the roots and 
quantification of the inorganic elements after dissolution of the residues 
in acid solution. 



9.2.3 Dispersion of the Particles 



Structure of the Soil and Organic Components 

As mentioned in the introductory section to this chapter, soil always 
contains varying proportions of coarse materials of inorganic (coarse 
sands, gravels) or organic origin (plant fragments), in addition to 
structural aggregates whose form and stability vary with the type of soil. 

In active medium (mull), humification processes result in relatively 
large quantities of transformed organic matter: microbial metabolites with 
a rapid turnover (e.g. many polysaccharides), and very stable phenolic 
products, both of which are accounted for by SOM decomposition 
models (Pansu et al. 2004). Both types of materials can bond to mineral 
matter to form organomineral complexes such as the cements contained 
in soil micro-aggregates. These micro-aggregates also comprise the 
building materials of larger aggregates containing organic particles, 
organic debris and microbial species (Fig. 9.1). 

In soils with a low level of activity (rnoder, mor), the formation of a 
strongly differentiated profile with a resistant organic matter horizon 
(rnoder, mor) is likely, along with a horizon in which redistributed 
organic matter accumulates resulting in the organomineral complexes of 
the structures of precipitation (Bruckert, 1994). 

Fractionation thus depends on the different forces of cohesion of the 
soil structure. In certain cases, simple moistening is enough to break 
down the macro-aggregates and disperse the fine particles (slaking). In 
other cases, more energetic dispersion techniques are needed to release 
the micro-aggregates and the organic fragments embedded in the 
structural aggregates. 



Physical Fractionation of Organic Matter 297 

Dispersion Techniques 

Dispersion consists in breaking certain organomineral binding forces 
without fragmenting plant debris, and if possible avoiding damaging 
microbial cells or the structure of the micro-aggregates. It should be kept 
in mind that the aim of granulometric fractionation of organic matters is 
very different from particle-size analysis of soil (cf. Chap. 2). In 
particle-size analysis, very energetic methods are used to destroy clay- 
humic "cements" e.g. destroying organic matter with hydrogen peroxide, 
destroying organomineral bonds with reagents that are highly complex- 
ing for iron and aluminium such as sodium tetraborate or sodium pyro- 
phosphate. 

Such techniques are not appropriate here since the organic components 
need to be recovered without them being damaged or dissolved. The most 
useful methods can be classified in three groups: 

- dispersion with water and possibly with mechanical agitation of varying 
strength 

- sonic and ultrasonic dispersion 

- chemical dispersion with dispersing reagents that are not too aggressive 
for organic matter. 

Mechanical Dispersion with Water 

Bruckert (1994) recommended this type of dispersion technique rather 
than techniques using ultrasounds whose action varies considerably with 
the type of organomineral cement of the aggregates and is considered to 
be too destructive for some soil compounds as ultrasounds break fragile 
minerals and damage certain organic matter, but especially "cause the 
breakdown of the microbial cells from which the protoplasmic contents 
come to be adsorbed on clays (Mc Gill et al. 1975)". 

The technique of Bruckert et al. (1978) is a low-impact mechanical 
treatment by controlled agitation in the presence of agate balls (35 g of 
dry soil sample from the fine earth prepared at 2 mm, 200 mL water is 
placed in a rotary shaker with five agate balls and agitated at 50 rpm for 
15 h). Feller (1979) developed a similar technique on tropical sandy soils 
with low humus content. In this case the recommended mechanical action 
is even more moderate: 100 g soil agitated for one hour with three glass 
balls in 300 mL distilled water. 

Andreux et al. (1980) studied a standard steppe soil of the chernozem 
type with a very stable clay-humus complex. The dry soil was sieved to 2 
mm, shaken by slow rotation (40 rpm) in water (35 g of soil for 200 mL 
water) for one night at 20°C with different numbers of agate balls. The 
rates of the fine clay-silt fraction (0-50 |Lim) obtained by these authors 
increased from 57% of the soil weight with agitation without agate balls 



298 



Organic Analysis 



to 90% of the soil weight when mechanical fragmentation was used. 
Beyond two, the number of balls had a limited influence on the rate of the 
fine fraction (Fig. 9.3). On the other hand, up to 15 h of agitation the rate 
increased without reaching the next stage, revealing that destruction of all 
aggregates bigger than 50 |um is progressive. However, beyond a certain 
degree of mechanical action (more than three balls for 15 h of agitation or 
five balls for more than 8 h of agitation), the treatments appear to 
solubilize part of the carbon, so very aggressive mechanical action is not 
recommended. 




Fig. 9.3. Influence of the number of 10 mm agate balls (a), and length of agitation 
(b) on the fragmentation of the aggregates >50 |im (after Andreux et al. 
1980) filled diamond mass percent of the 0-50 |im fraction, filled square 
carbon content of the 0-50 |im fraction as a percentage of total C 

Sidi (1987) also used fragmentation by agitation with glass balls on a 
Tunisian carbonated soil. Fig. 9.4a shows the influence of the length of 
agitation on particle size distribution with or without the presence of 
three glass balls (15 g of soil, 100 mL water, back and forth agitation 
with one back and forth movement per second. The main effect of the 
mechanical treatment was the destruction of the biggest macro-aggregates 
(200-2000 |um) whereas the percentage of aggregates of intermediate 
size (50-200 |um) remained almost identical with or without the balls. 
The shape of the curves also suggested that the process of fragmentation 
occurs in stages (1) division of the biggest aggregates (>200 |um) into 
intermediate aggregates (50-200 |um) during the first 30 min of agitation, 
followed by (2) division of the 50-200 |um aggregates into micro- 
aggregates of the size of clays and silts (0-50 urn). In contrast to the 
situation illustrated in Fig. 9.3, one hour of agitation with three glass balls 
was enough to reach a dispersion plateau for this type of soil. 

Monnier et al. (1962) recommended performing dispersion pro- 
cedures before densimetric fractionations (cf Sect. 9.2.4): either by dry 



Physical Fractionation of Organic Matter 



299 



sieving to 500 um, or boiling in water followed by one rinse in alcohol 
and one period in the drying oven. 





4 6 

Ultrasound time min 



Fig. 9.4. Dispersion of a Mediterranean soil with water (according to Sidi 1987, 
15g soil/150 ml_ water, back and forth shaking apparatus, 1 backwards- 
forwards movement per second) a: influence of length of agitation with 
and without three glass balls filled square, open square 200-2,000 |im 
with and without balls, respectively, filled triangle, open triangle 50-200 
|im with and without balls, filled circle, open circle 0-50 jim with and 
without balls; b: influence of ultrasounds (80 W-80 kHz) on agitation for 
1 h without balls 



Sonic and Ultrasonic Dispersion 

Although occasionally severely criticized for being too destructive for 
certain organic matter (Bruckert 1994), sonic and ultrasonic dispersion 
techniques are generally recommended for the physical fractionation of 
soil organic matter. 

Edwards and Bremner (1967) subjected an aqueous suspension of the 
soil sample (10 g soil for 25 mL water) to sonic vibration (9 kHz, 50 W) 
with a Raytheon S-102A vibrator (Raytheon Co., Norwood, MA USA). 
For 14 soils of very different texture, dispersion in fine particles of the 
size of clays (<2 urn s) by sonic vibration for 30 min was evaluated by 
the pipette particle-size fractionation method (cf. Chap. 9). Fig. 9.5 
shows that dispersion was always much higher than by simple agitation 
in water. Dispersion was comparable with that obtained with the two 
chemical dispersants tested: calgon peroxide and sodium resin. 

The rate of dispersion obtained on the suspensions with an ultra- 
sonic probe MSE Cabinet Model 60 Ultrasonic disintegrator (Measuring 
and Scientific Equipment Ltd, London) delivering a frequency of 18-20 



300 



Organic Analysis 



kHz and a power of 60 W, is very similar to that obtained by sonic 
vibration (Fig. 9.6). Beyond 30 min (the period recommended by the 
authors) the duration of sonification had only a slight influence on the 
percentage of clay obtained (Fig. 9.7), Fig. 9.4b shows a comparison of 
the influence of the period of sonification observed by Sidi (1987) with a 
slightly more powerful high frequency ultrasound probe (80 kHz, 80 W); 
in this case a plateau was reached earlier (at 8-10 min) for the dispersion 
of the particles the size of silt (0-50 |um). 



90 
80 



70 

1 60 



E 50 

c\i 40 
o 



C/) 
C/) 
03 



30 

20 

10 





-- 


—o— Shaking in water 

-■- Ultrasounds 

A Calgon-hydrogen 
peroxide 

-x- Na resin 




-- 


1 1 1 1 1 1 r 





10 15 

Soil test sample 



Fig. 9.5. Rates of clayey fractions obtained by ultrasonification and three other 
dispersion techniques on 14 soils (after Edwards and Bremner 1967) 



After a detailed analysis by Watson (1971) of the ultrasonic vibration 
method applied to the dispersion of soils, Genrich and Bremner (1972) 
re-evaluated the technique following some criticism of its use. They used 
28 soils covering a very varied field of pH (3.6-8.2), carbonate content 
(0-34% CaC0 3 , texture (2-59% of sand, 7-72% of clay) and organic 
content (0.14-9.4% organic C). They tested two types of instruments 
(Heat Systems Ultrasonics Inc, Plainview, NY USA) (i) a standard 
Branson W-185C model with probe (20 kHz, 80W) and (ii) a Branson 
220 ultrasonic cleaner with stainless steel tank (40 kHz, 100W). Different 
procedures were used with the tank model (soil: water ratios of the 
suspensions, sonification in an Erlenmeyer flask or directly in the 



Physical Fractionation of Organic Matter 



301 



tank). With the probe model, the end of the probe (diameter 1.27 cm) was 
immersed to 2 cm below the surface of the suspension (10 g of soil in 25 
mL water) in tubes of steel cooled on the outside to less than 20°C. 



Fig. 9.6. Comparison of clay rates 
obtained on five soils by 
sonic (9 kHz, 50 W) and 
ultrasonic (18-20 kHz, 60 W ) 
dispersion for 30 min on 
suspensions of 10 g of 
sample in 25 mL water (after 
Edwards and Bremner 1967). 




In all cases, more complete dispersion was obtained with the probe 
model than with the tank model. However, the dispersion provided by the 
probe depended to a great extent on the quality of its surface: with a 
pitted probe, the authors observed that the length of time needed for 
dispersion was two to four times longer than with a probe in good 
condition. It is consequently important to gently polish the end of 
the probe with a fine abrasive paper after each 30-min period of use. 
According to Genrich and Bremner, the imperfect condition of the probes 
could explain the failures noted by other authors before their trials. 

They also showed that with a 15-min period of sonification with the 
probe, under the conditions described above, the clay rates obtained on 
their 28 soils were always equal to or higher than those obtained with the 
sodium peroxide and polyphosphate method of Kilmer and Alexander 
(1949) which at that time, was the standard method of dispersion (Soil 
Survey, 1960). This study clearly demonstrated the dispersion power of 
ultrasonic probes. However, the authors' conclusion was cautious saying 
that no method of dispersion can be described as universally applicable 
for all soils. 

Anderson et al. (1981) studied the distribution of organic matter in the 
particle fractions of two soils of the chernozem type. They carried out the 
dispersion of these soils by ultrasonic vibrations for 8 min with more 
power than previously (300 W, apparatus Bransonic 1510) but applied to 
more diluted suspensions (soil: water ratio of 1:10). Tiessen and Stewart 
(1983) studied the effect of cultivation on the organic composition of the 
particle fractions using a procedure similar to that of Anderson et al. 
(1981). 



302 



Organic Analysis 



On a soil of the chernozem type, Shaymukhametov et al. (1984), like 
Anderson et al. (1981), observed a stage of fragmentation of micro- 
aggregates (<50 |um) after sonification for 30 min, whereas in one 
minute, 96.4% of the larger aggregates were destroyed. Their experiment 
highlighted the very great difference in stability between micro- 
aggregates and structural aggregates (Fig. 9.1). It should also be noted 
(Fig. 9.7) that the degree of stability of the three sizes of micro- 
aggregates between 1 and 50 urn, is very similar, the probable 
explanation being that ultrasounds cause the progressive release of fine 
clayey particles from the three classes of the silt-size micro-aggregates 
with no distinction between the classes. This is different from the 
behaviour of structural aggregates where there is a clear difference in 
stability between the 50-200 |um and 0-50 |um fractions (Fig. 9.4). 




Fig. 9.7. Effect of the duration of ultrasound treatment on fragmentation of micro- 
aggregates <50 |im (after Shaymukhametov et al. 1984): filled square 
<1 |im, filled diamond 1-5|im, filled triangle 5-10 |im, times 10-50 |im 

In order to study the organic matter of an aquoll, Catroux and 
Schnitzer (1987) performed ultrasonic dispersion on soil in water 
suspensions at a ratio of 1:5 (between the ratios used by Genrich and 
Bremner, 1972, and by Anderson et al. 1981). 100 g of soil in 500 mL 
distilled water were agitated on a magnetic stirrer and treated by 
ultrasound with a Blackstone SS2 generator. Power was applied at 
400 W for 15 min, (which is a more energetic treatment than that applied 
by the preceding authors) and the end of the probe was immersed to 2-3 
cm below the surface of the liquid in order to decrease the swirling 
action. 



Physical Fractionation of Organic Matter 303 

Gregorich et al. (1988) tried to define and quantify the action of 
ultrasounds more precisely. Ultrasonic vibrations cause cavitation due to 
the formation of microscopic bubbles resulting from local reductions in 
pressure and the subsequent bursting of these bubbles. When the bubbles 
burst in the suspension, they produced waves of pressure of sufficient 
mechanical energy to break the aggregation bonds. These authors used a 
20 kHz Branson probe whose power could be adjusted from to 150 W. 
The probe head (diameter 12 mm) was immersed to between 5 and 10 
mm below the surface of the suspensions (15 g of 1-2 mm aggregates in 
75 mL water). The output power of the probe was gauged by measuring 
the rise in temperature of a known water mass over a given period. 
Gregorich et al. considered that the most significant parameter is the 
quantity of energy applied per mL of suspension: 

J=PtV l 

where J is the applied energy in J mL" 1 , Pis the output power of the 
probe in W, t is the time in s, Fis the volume of suspension in mL. 

Figure 9.8 shows the results obtained by these authors on a melanic 
humus gley horizon of a cultivated brunisol. This type of material has 
very resistant silt particles. None of the ultrasonic treatments enabled 
their fractionation as thoroughly as treatment by agitation in the presence 
of hydrogen peroxide. The principal bond between these particles thus 
appears to be primarily organic. These authors also observed stronger 
bonds between macro-aggregates (or pseudo-sands) than in the majority 
of studies quoted above, energy ranging between 300 and 500 J mL" 1 
being required to disperse these aggregates which are relatively rich in 
organic matter. As is true for silt particles, organic matter thus seems to 
act as cement, particularly in macro-aggregates. One possible explanation 
is that their organic functioning is a little different (partly anaerobic) in 
this type of soil from the examples above. 

Balesdent et al. (1991) studied the effect of ultrasounds on the 
granulometric distribution of the organic matter contained in 17 soils (Ap 
horizons of cultivated soils), type brown soils, or not very processed 
alluvial soils. The procedure they used combined mechanical and 
ultrasonic dispersion techniques. The first mechanical dispersion used 
rotary shaking of the aqueous suspensions with glass balls similar to the 
techniques described above (Andreux et al. 1980). Sonification was then 
applied to the fraction below 50 or 25 |um in order to split it into three 



304 



Organic Analysis 



particle sizes: 0-0.2, 0.2-2, 2-50 |um (or 2-25 um). The ultrasound 
apparatus used was the same type as the one used above (Branson cell 
disintegrator, 20 kHz, 150 kW, probe with a flat head 13 mm in 
diameter). 







o 



o 



500 



2-50 H?0 



2^2 



>50 H 2 2 




<2 HoO 



2^2 



500 



1000 



1500 



Applied energy J mi- 



Fig. 9.8. Ultrasonic dispersion of a melanic humus gley horizon of a cultivated 
brunisol (after Gregorich et al. 1988): filled triangle >50 jam fraction, 
filled square 2-50 |im fraction, filled diamond <2 |im fraction; horizontal 
lines represent dispersions obtained after H 2 2 treatment of destruction 
of organic matter 



A kinetic study of the action of ultrasounds on silt-size micro- 
aggregates was performed by Balesdent et al. (1991). Sonification was 
applied to suspensions of 100 mL with a soikwater ratio of 1:3, the probe 
was immersed to 3 cm below the surface and the apparatus regulated at 
70% of its power (corresponding to 0.5 W mL - l from the manufacturer). 
Figure 9.9 shows changes in 0-0.2 and 0.2-2 |um fractions and their sum 
(0-2 |Lim) compared to the reference method (hydrogen peroxide treat- 
ment and pyrophosphate dispersion). 

Compared to the results of Gregorich et al. (Fig. 9.8), the dispersion of 
soils studied by Balesdent et al. appears to be easier as it has a stable 
production of 0-2 urn clay fraction from 300 to 1,800 J mL -1 (Fig. 9.9). 
In comparison, the clay fraction in Fig. 9.8 there is a continuous increase 
with an increase in the energy applied. However, Fig. 9.8 shows a clean 
break in the slope of the surve of the clay fraction for an energy 
of approximately 300 J mL -1 , i.e. about the energy needed to reach the 
stage shown in Fig. 9.9. In a clay latosol, Roscoe et al. (2000) found that 



Physical Fractionation of Organic Matter 



305 



energies of 260-275 JmL -1 were sufficient to break down unstable 
aggregates (2,000-100 |Lim) and to leave stable aggregates (100-2 |Lim) 
unchanged. 




600 



1200 i 



Applied energy J ml_ 




t r 

1200 1800 



Fig. 9.9. Effect of the energy of the ultrasonic treatment on fragmentation of 
micro-aggregates the size of clays in an alluvial soil (1) and a 
weathered brown soil (2); horizontal lines represent dispersions 
obtained by chemical treatment with H 2 2 then Na 3 P0 4 (Balesdent 
et al. 1991): filled square 0-0.2 jim fraction, open circle 0.2-2 jim 
fraction, closed circle 0-2 jim fraction 

It is difficult to compare the data of Balesdent et al. (Fig. 9.9) with that 
of Shaymukhametov et al. (Fig. 9.7) where the cutting threshold of the 
fine fractions was at 1 urn. However, in both cases, the length of 
sonification needed to reach the 0-2 |um and 0-1 |um stages was quite 
similar. The more detailed study by Balesdent et al. of the fractionation of 
the fine 0-2 |um fraction provided interesting additional information on 
two aspects: 

- even with the highest energy of sonification, a stable stage is not reached 
for the fine fraction below 0.2 urn, and the dispersion of this fraction is 
always lower than that obtained with the chemical method of reference; 

- on one of the soils, the intermediate fraction (0.2-2 urn) reached 
maximum after around 5 min of sonification (150 J mL~l). This 
suggests an initial stage in the fragmentation represented by the 
division of the aggregates of silt size (2-25 |um) into smaller units (0.2- 
2 |um) rather than the fragmentation of the 0.2-2 |um fraction. The 
behaviour of associations within the clay-size fraction is apparently 
different from that observed within the silt-size fraction where the three 
particle-size ranges studied (Fig. 9.7) displayed the same stability. 
Instead it resembles that observed for macro-aggregates (Fig. 9.4): the 



306 Organic Analysis 

large structural aggregates (>200 |um) are less stable than the 
intermediate macro-aggregates (50-200 |um). 
Finally, for the soils they studied, Balesdent et al. recommended a 

sonification period of 10 min (600 J mL" 1 ) in the conditions described 
above. Dispersion of the silt micro-aggregates (to 2 |um) can then be 
considered complete, whereas the coarse clay fraction (0.2-2 |um) must 
be considered as micro-aggregated. 

Balesdent et al. also studied the effect of ultrasounds on the coarse 
organic debris separated in water after the action of glass balls. The study 
was on an alluvial soil containing 27% clay, and 0.9% organic carbon 
with a pH of 7. Corn and maize had been grown on the soil for 17 years 
so the coarse fragments mainly came from these plants. The ultrasound 
treatment was applied at different energies to aqueous suspensions at a 
ratio of 1:200 of each of the three light fractions: 200-2,000 |im ? 50-200 
|iim and 25-50 |um. The suspensions were then sieved at 25 |um and if 
necessary at 50 and 200 |um. The 0-25 |um suspension was separated by 
sedimentation into fractions of 0-5 |um and 5-25 |um. 

The results showed a very destructive effect of ultrasounds on the 
organic debris. After 10 min (the recommended time for fractionation of 
clayey particles), more than 60% of the carbon of the initial coarse 
organic fraction was split into the lower particle-size ranges, and this was 
the case for each of the particle-size ranges studied. These authors 
showed that part of this fractionation results from the separation of clay 
fractions associated with plant fragments; but cleaning of the plant 
fragments is insufficient to explain the quantities of organic matter 
transferred to the finer fractions. 

The use of ultrasounds under the conditions required for dispersion of 
clays produces marked fractionation of the coarse plant fragments. This 
significant observation led the authors to propose a procedure for particle 
size fractionation that uses only ultrasounds for the suspension of 
particles of less than 50 |um (cf. Sect. 9.2.4). 

Chemical Dispersion 

Chemical dispersion techniques are less widely used for organic 
fractionation than for classical soil particle size analysis (cf. Chap. 2). 

As mentioned in Sect. 9.2.2, sequestering reagents such as sodium 
tetraborate or hexametaphosphate can only be used to disperse clay 
soils when the aim is to recover roots or coarse plant fragments 
(Anderson and Ingram, 1989). But even in this case, there is a risk of 



Physical Fractionation of Organic Matter 307 

discolouration that subsequently makes it difficult to identify living roots, 
and of modification of the organic contents. 

Dispersing reagents that are highly destructive for organomineral 
bonds are not recommended for the study of particle-size distribution of 
organic matter. Their too high extracting power, in particular of humic 
and fulvic acids (cf Chap. 11), is likely to distort the results of such 
studies. Less aggressive extracting reagents should be used such as 
monovalent neutral salts which cause the dispersion of clays by exchange 
with the di- or trivalent cations of the exchange complex and the conse- 
cutive rupture of certain organomineral links. 

Ladd et al. (1977), Oudinot (1985), Sallih and Pansu (1993) used a 
sodium bicarbonate solution as a complement to the mechanical action of 
agitation for the initial dispersion of the soils. 

Sodic resins have also been used for the dispersion of soils (Edwards 
and Bremner 1967, Rouiller et al. 1972). Adapted from studies by 
Edwards and Bremner, Fig. 5 shows that using the resin technique, 
dispersion is slightly higher than using the two other methods tested for 
most of the 14 soils in this experiment. 

Feller et al. (1991) compared different dispersion techniques, including 
an IRN77 amberlite resin in a sodic state. The resin was tested alone (R) 
or combined with ultrasonic fractionation on the fraction below 50 |um 
(R/US). The resin technique was compared with five other dispersion 
methods which were all combined with the same ultrasonic fractionation 
of the fractions below 50 |im: a B/US method similar to that of Balesdent 
et al. (1991) described below, an NaCl/US method replacing the water in 
the suspensions by M sodium chloride solution, an M sodium hydroxide 
method bringing the suspension to pHIO (pHIO/ US) and a 3.3 g L _1 
sodium hexametaphosphate method (HMP/US). 

Figure 9.10 shows the comparative effectiveness of the different 
methods on a ferrallitic soil from Martinique. Up to the level of fine silts, 
the most effective dispersion was obtained using the R/US technique 
(resin on the total soil then ultrasounds on the fraction below 50 \xm). On 
average, solubilization of organic matter was less than 4% of total carbon 
in the 19 soils studied. Based on the results of this experiment, the R/US 
technique appears to be preferable to the technique using glass balls plus 
ultrasounds (B/US) described above (Balesdent et al. 1991). However, 
the two methods were not tested on the same types of soils. In addition, 
the authors mentioned practical constraints in the use of the resins: the 
time needed for resin regeneration and preparation is rather long and 
there is a risk of contamination of the soil by very fine resin (<50 \xm). 



308 



Organic Analysis 



500 



T 400 

a> 

E 300 



200 
100 





■Mil 






O 

? 

O 

s 



en 
O 

o 
o 



O 
I 

o 



O 



o 

! 



o 
ho 
I 

M 



fraction, jim 






DB/US 

□ NaCI/US 

□ pH10/US 
DHMP/US 
DB/R 

□ R 

■ R/US 



Fig. 9.10. Effect of different dispersion methods on particle size fractionation of 
a ferrallitic soil from Martinique (according to Feller et al. 1991): 
US: ultrasonification of the 0-50 jim fraction, B: stirring with balls, 
NaCI: NaCI solution, pH10: NaOH solution, HMP: sodium hexameta- 
phosphate solution, R: stirring with sodic resin 

The sodic resin technique was also shown to be the most effective of 
the five dispersion techniques for stable oxisols with high gibbsite 
content (Bartoli et al. 1991). These authors also studied the influence of 
the soil:sodic resin ratio on dispersion, pH of the suspensions, and carbon 
solubilization. Volumes of 0, 10, 50, 100, 200, 300, 400 mL Amberlite 
IR-120 (500 |um) sodic resin in nylon bags with a 50 urn mesh were 
added to samples of 2.5 g soil in 200 mL distilled water. The suspensions 
were agitated for 16 h on a rotary shaker at 40 rotations per minute. The 
results in Fig. 9.11 show a stable stage of aggregate breakdown for 
volumes of resin ranging between 50 and 200 mL, this corresponds to the 
volume (100 mL) used by Feller et al. (1991). There was a rise of 
between one and two units in the pH of the suspensions; in all cases the 
final pH remained lower than that of the main extracting reagents of the 
humic acids (cf Chap. 11). In the deeper horizon, dissolution of organic 
carbon only became perceptible with volumes of resin above 200 mL; on 
the other hand, in the cultivated surface horizon, the authors noted 
dissolution of organic carbon at all doses of resin independently of the 



Physical Fractionation of Organic Matter 



309 



added volumes; this horizon probably contains more recently formed 
organic matter which is not very humified, and is water soluble. 



100 

^ « 

<2jim 




> 50 ^m 
2-20 jam 




> 50 urn 
i i3 2-20 jim 



0.2 



0.4 



0.2 



0.4 





0.4 

Volume of Na* resin (L) 

Fig. 9.11. Influence of the volume of sodic resin (2.5 g for 200 ml_ distilled water) on 
dispersion of the aggregates, the pH of the soil suspension, and 
solubilization of organic carbon in a surface horizon (on the left) and a 
deep horizon (on the right) of a Nigerian oxisol (Bartoli et al. 1991) 



9.2.4 Separation by Density 



The Techniques 

The first methods used to separate the organic fragments in the soil were 
usually based on an obvious physical property: the density of free organic 
matter, which is close to 1, is lower than that of the organomineral 
complex. However, the first density techniques were used for the 
separation of primary minerals (Pearson and Truog, 1937). Starting from 
the work of Lein (1940), Henin and Turc (1949) adapted a densimetric 
separation technique for free organic matter in soils. Fractionation was 
performed in beakers containing a mixture of bromoforme and benzene. 



310 Organic Analysis 

The technique was improved by Jeanson-Luusinang (1960) by the use of 
special decantation funnels, and then further improved by Monnier et al. 
(1962) who adapted an earlier technique of Lein (1940) for density 
separation by centrifugation. 

The centrifugation technique improved the use of differences in 
density, but neither of these two techniques extracts all the light organic 
matter. Monnier et al. (1962) carried out tests with synthetic mixtures of 
mineral soil and oat straw. For fragmentation of straw in particles of less 
than 0.2 mm, the recovery rate was 73% of the added straw with the 
technique of Monnier et al. and 44% of the added straw with the 
technique of Jeanson-Luusinang. The method of Monnier et al. was used 
to model the evolution of carbon stocks by Arrouays (1994). 

Greenland and Ford (1964) used ultrasounds to disperse the aggregates 
before density separations (cf sect. 9.2.3). The technique was improved 
by Ford et al. (1969) with the use of surfactant and of dibromochloro- 
propane (DBCP density = 2.06) instead of bromoforme for density 
separations. At that time authors were not concerned with the possible 
toxicity of these products, which today is widely acknowledged. 

Turchenek and Oades (1979) studied methods of density fractionation 
of organic matter by combining them with preliminary particle-size 
fractionations. They carried out from 4 to 7 density fractionations 
with mixtures using decalin (decahydro naphthalene d = 0.88), 
dibromochloropropane (DBCP d = 2.06) and bromoforme (d = 2.88) on 
most of the seven standard particle ranges (coarse sands, fine sands, 
coarse silts, fine silts, coarse clays, medium clays, fine clays). 

Their observations showed that more than 50% of the light fraction 
(d < 2.06) with a particle size of coarse and fine sands is made up of 
organic matter. The fraction comprising coarser particles is mainly made 
up of recognizable plant fragments with high C:N ratios and low 
solubility. The fraction made up of finer particles (fine sands to coarse 
clays) contains a higher proportion of identifiable microbial cellular 
debris and soluble aromatic humic compounds. 

The light clay fractions are also rich in organic materials. Forms of 
oxidized iron, aluminium and silicon are present to a significant degree in 
all the fractions, indicating a wide range of different interactions between 
inorganic and organic matter. 

Nowadays none of the density methods using chlorinated heavy 
solvents are used because of the toxicity of this type of solvent and of the 
safety requirements in laboratories. 

Dabin (1976) proposed a method for the fractionation of organic 
materials (cf. Chap. 11). The first part of this method comprised density 



Physical Fractionation of Organic Matter 3 1 1 

fractionation on phosphoric acid 2 M ( d = 1.2). In addition to its low 
toxicity compared to density liquors, this type of acid treatment has the 
advantage of destroying carbonates in calcareous soils releasing a certain 
proportion of sequestered plant material. Sidi (1987) used this technique 
to separate light fractions from mixtures of soils and wheat straw 
incubated in controlled laboratory conditions. The method was used 
to propose a descriptive model of carbon dynamics with three 
compartments (Pansu and Sidi 1987). 

The Ladd et al. (1977) method includes a series of particle-size and 
density fractionations that are also suitable for calcareous soils. A 
modification of this method allowed, in its first stage, fractionation of the 
light materials from in vitro incubation experiments of mixtures of soils 
and 14 C labelled wheat straw (Cortez 1989, Sallih and Pansu 1993). The 
suggested modification concerned the use of an aqueous saturated zinc 
sulphate solution (d = 1.4) as heavy liquid, whereas Ladd et al. had used 
carbon tetrachloride (d = 1.59). Among the different high-density 
saturated saline solutions possible, zinc sulphate and ferrous sulphate 
(density = 1.6) appeared to be particularly promising. A zinc sulphate 
solution was selected to avoid the sequestering of iron on the 
organomineral complexes. However, it is probable that the zinc element 
also results in the formation of certain complexes. Other mineral density 
liquors have also been used including zinc chloride solutions (Besnard 
et al. 1996), sodium metatungstate (Elliott et al. 1991), sodium 
polytungstate (Cambardella and Elliott 1993, Golchin et al. 1994, Six 
et al. 1999), Ludox, aqueous suspension of silica colloidal particles 
(Meijboom et al. 1995). 

Anderson and Ingram (1989) recommended methods of fractionation 
of light materials with water that are rather similar to those described for 
the extraction of roots. The light fraction is defined as the fraction (1) 
which floats when it is dispersed in water, (2) which passes through a 
sieve of 2 mm but not through a sieve of 0.25 mm. However these 
authors pointed out that elutriation and sieving methods separate 
significantly less free organic matter than density methods. The methods 
of separation in water are nevertheless worthwhile because less organic 
matter is solubilized in water than in dense liquors, which are often rather 
corrosive (Beare et al. 1994, Puget et al. 1996). 



312 Organic Analysis 

Procedures 

Only procedures for the methods of Monnier et al. (1962), Dabin (1976) 
and density liquor ZnS0 4 are described here (cf. Sect. 2.4.1 "The 
Techniques" for modifications). 

Method Using Organic Heavy Liquid 

The density liquid should be adjusted to the density selected by mixing 
bromoforme with a lighter solvent, preferably alcohol (Monnier 
et al. 1962). The density recommended by the authors who worked on 
silt soils in the area of Versailles (France) is 2. These authors pointed 
out that more complete separation could be achieved by the successive 
use liquids of density 1.75, 2 and 2.25. 

The soil sample should be air dried and crushed to 2 mm particle size. 
Weigh 5-10 g of soil depending on the free organic matter content. The 
weight of the sample can be also adjusted as a function of the techniques 
to be used for quantification after fractionation (e.g. carbon determi- 
nation on the whole light fraction). Place the sample in the 100 mL tube 
of a centrifuge, and fill the tube with the density liquid. After stirring 
with a glass rod, centrifuge for 5 min with an acceleration of about 
l,000g in the centre of the tube. Collect the supernatant on a flat filter and 
repeat the operation again by suspending the centrifugation pellet in the 
heavy liquid. 

It is possible to destroy aggregates to release embedded light organic 
matters before fractionation either by boiling in water then washing with 
alcohol and drying in the drying oven, or by sieving the dry sample at 
500 |um. 

In the case of soils with high free organic matter content, the risk of 
sequestration of dense particles within light organic materials is high and 
it is thus recommended to centrifuge the light materials again after 
washing in a different tube with the heavy liquid. 

Density Method with Phosphoric Acid 

The method of Dabin (1976) applies to soil sieved to 0.5 mm. The weight 
of the sample can vary from 5 g to 40 g depending on the organic content. 
Agitate for 30 min on a back and forth shaker (1 backwards-forwards 
movement per second) with 200 mL of a 2 M phosphoric acid aqueous 
solution (136 mL L" 1 ). Centrifuge for 20 min at 3,000g then transfer the 
supernatant on a filter. Repeat this operation twice. Dry and weigh the plant 
matter recovered on the filter. The total carbon of this material can be 
measured by combustion and determination of released carbon dioxide; 
nitrogen can be measured simultaneously with a CHN analyser, or 
separately using the Kjeldhal method (cf Chap. 10). 



Physical Fractionation of Organic Matter 



313 



Method by Sieving and Inorganic Heavy Liquid 

Figure 9.12 summarizes the procedure for extraction of free organic 
matter (FOM). Sieve a 80 g soil sample on a 5 mm sieve and put in 
suspension in 300 mL of 0.2 mol (NaHC0 3 ) L" 1 aqueous solution. After 
1 h of moderate agitation on a rotary shaker, centrifuge at 12,000g for 
30 min. Collect the light fraction by filtering the supernatant. The extrac- 
tion can be repeated twice. 





Test portion 




) 3 0.2 mol l_- 1 




80 g 


300 mL NaHCC 






Shaking 

centrifugation 

filtration 








ater 
/=1.4 




Watersoluble 


Centrifugation pellet 500 mL w 










Shaking 
sieving 50 |um 










nm 




<50jum 




>50 












ZnS0 4 solution c 
Shaking 
centrifugation 
filtration 










Free 

organic 

matter 






Sane 






J 










i 


r 







Continuation Fig 9.13 
Fig. 9.12. Diagram of the separation of free organic matter by density and sieving 

Suspend the centrifugation pellet in 500 mL water. For dispersion, 
place on a rotary stirrer for 2 min at maximum speed. Sieve on a 50 |um 
sieve in water. Suspend the coarse fraction (greater than 50 |um) in a 
heavy aqueous solution saturated in zinc sulphate (density 1.4). After 
30 min of agitation at mean velocity, centrifuge at 3,000g, then filtrate 
the supernatant on a 3 |um Millipore membrane. Wash the light fraction 
recovered on this filter carefully four times with water, add to the 
previous light fraction; dry in the drying oven at 30°C. 



314 Organic Analysis 

If the sands do not have to be separated from FOM, the method can be 
simplified by leaving out density separation on the coarse fraction. In this 
case FOM is estimated by carbon determination on the fraction: "light 
matter separated on NaHC0 3 + fraction greater than 50 |Lim". Certain 
types of soils studies with 14 C tracers have shown that this simplified 
estimate is significantly more exhaustive than the preceding one (Sallih 
and Bottner, Cefe-CNRS Montpellier, France, unpublished data). 

9.2.5 Particle Size Fractionations 



Limits of Density Methods 

The use of the density method on its own for the study of organic 
components has sometimes been criticized, but not when density 
fractionation was coupled with particle-size fractionation. One of the 
reasons mentioned above is the toxicity of heavy organic liquids. This 
obstacle can be overcome by using heavy aqueous solutions saturated 
with mineral salts. According to Bruckert (1994), using density as the 
only criterion can also be challenged for several reasons: 

- the ideal density to use varies with the type of soils. Thus, with a 
density of 1.8, 90% of the organic matter of andosols can be separated 
in the light fraction whereas in brown soils the percentage is only 20% 

-the density of the plant debris increases during decomposition by 
incorporation of mineral matter which can be determined by ash 
quantification 

- in the case of organic heavy liquids, organic compounds can fix on 
clays and perturb subsequent studies. As mentioned earlier, inorganic 
heavy liquids do not have this disadvantage, but they can modify 
organomineral complexes. 

Procedures for Particle-Size Fractionation 

Given the remarks quoted in Sect. 9.2.3 about aggregate dispersion, it is 
difficult to describe a single procedure for particle size fractionation for 
all soil types. However four procedures appear to be appropriate for 
different soil types: 

- the continuation of Section "Methods by Sieving and Inorganic Heavy 
Liquid" adapted from Ladd et al. (1977) on calcareous soils 

- Agitation with Glass Balls and Ultrasonification (Balesdent et al. 
1991) used on different cultivated soils of France 



Physical Fractionation of Organic Matter 



315 



"Resin H + and Ultrasounds" (Feller et al. 1991) used on tropical soils 
of various origins, with a simplified alternative for sedimentation 
(Gavinelli et al. 1995) 
a special procedure for use on sandy soils (Feller 1979; Feller et al. 1991). 



Continuation of the Procedure Described in "Method by Sieving 
and Inorganic Heavy Liquid" (cf. Sect. 9.2.4) 

In addition to separation of the "free organic matter" fraction described in 
"Method by Sieving and Inorganic Heavy Liquid" (Fig. 9.12), this 
method provides: 

- a water-soluble organomineral fraction 

- a fraction of more than 50 urn (primarily inorganic, density >1.4) 

- an organomineral fraction with particles of less than 50 urn. 

Cf. Fig. 9.12 



<50 jam 
fraction 



Centrifugation 
15min at 4 000 g 
(2 times) 



<0.2 |im 
fraction 



i 



Centrifugation pellet 
50-0.2 urn 



Dispersion in water 
centrifugation at 800g (2 times) 



50-2 urn 
fraction 



Centrifugation pellet 
Drying 



Surpernatant 
Evaporation -drying 



2-0.2 urn 
fraction 



Fig. 9.13. Fractionation by centrifugation of the clay-silt fraction (complement of 
Fig. 9.12, after Ladd et al.1977) 

The complete method includes the separation of this last fraction into 
particles the size of silts (2-50 urn), coarse clays (0.2-2 |um) and fine 
clays (0-0.2 um). The separation procedure described by Ladd et al. 
(1977) shown in schematic form in Fig. 13 should be used: (1) centri- 
fugation for 15 min at 4,000g in a 250 mL tube makes it possible to 
separate the fine fraction (less than 0.2 |um) in the supernatant; (2) the 
centrifugation pellet is then suspended again with water and centrifuged 



316 Organic Analysis 

for 5 min at a low speed (800g); repeated twice, this operation makes it 
possible to isolate a centrifugation pellet of silt size (50-2 |um) and (3) a 
supernatant of coarse clay size (0.2-2 |um). 

The two clay fractions and the water-soluble fraction are concentrated 
in a vacuum rotary evaporator at 40°C. The method (Figs. 9.12 and 9.13) 
thus provides six fractions: a water-soluble 0-0.2 |um fraction, a 0.2- 
2 |um fraction, a 2-50 |um fraction, a heavy coarse fraction (size > 50 |um 
and density > 1.4), and a light coarse fraction (size > 50 |um and density 
< 1.4). 

Dry each fraction in a Petri dish at a low temperature, depending on 
subsequent measurements either at room temperature (light matter) or in a 
drying oven or sand bath. 

Agitation with Glass Balls and Ultrasonification 

Figure 9.14 is synoptic diagram of fractionation after Balesdent et al. 
(1991). Dry the soils in air and sieve to 2 mm using a grinding-sieving 
machine with rollers (Pansu et al. 2001). Put a 50 g sample in a 250 mL 
plastic bottle with 180 mL of water and ten glass balls 5 mm in diameter. 
Agitate the bottle on a rotary shaker at 40 rpm for 16 h. 

Filter the suspension underwater on a sieve with a 200-|um square 
mesh. Put the nib in suspension in a beaker. The organic fragments are 
separated during their transfer to a 200-|um sieve by decantation. Repeat 
this operation several times until the sands no longer contain any visible 
organic fragments. Perform the same operation on the fraction of less 
than 200 |um with a 50-|um sieve to obtain F200-2000, M200-2000, 
F50-200, M50-200 fractions (F being the organic fragments, M the org- 
anomineral part). 

Centrifuge the suspension with particles <50 \im to separate the 
particles of less than 0.2 |um (cf. Continuation of the Procedure Described 
in); reserve the supernatant. Suspend the centrifugation pellet at a 
solid:water weight ratio of approximately 1:3. Subject the suspension to 
ultrasound treatment for 10 min under the conditions described in above 
i.e. at an applied energy of approximately 300 J mL" 1 . In samples 
containing limestone, it is recommended to eliminate carbonates in the 
suspension after ultrasound treatment by adding HC1 solution to a pH of 
3.5 on the pH-meter, and to wash the solid residue before subsequent 
separations. 

The 2-50 |um, 0.2-2 |um and 0-0.2 |um fractions are separated by 
centrifugation techniques similar to those described earlier. The 
conditions chosen here are only slightly different from those of the 
previous authors: 25 min at 2,900g to separate the fine fraction <0.2 |um 
by decantation and 3 min at 800g for the 0.2-2 |um fraction. These 



Physical Fractionation of Organic Matter 



317 



conditions must be recomputed each time based on Stokes law as a 
function of the operating conditions (cf. Chap. 2). 



50 g soil 



10 glass balls 



250 mL 
suspension 



Organic debris 
200-2,000^m 



180 mL water 



Rotary shaking 16 h 

40 rpm 
sieving 200 iwm 



<200 jum 
fraction 



Decantation on 
200 (im sieve, 



Sieving 50|uim 



Coarse 
sands 



<50jum 



Centrifugation 



Organic debris 
50-200 urn 



Decantation on 
50|um sie ve 



I Sand 50-200 urn I 



|<0.2|um 



Flocculation 
CaCl2 
0.5 g IT 
Centrifugation 



-1 



1 0.2-50 urn 



Water soluble 
fraction 



0-0.2 urn 
fraction 



Ultrasounds 



-1 



10min30 J mL" 
Decarbonatation if necessary 
at pH 3.5 
Successive centrifugations 



0.2-2 nm 
fraction 



2-50 jam 
Mesh plus 



Fig. 9.14. Particle size and centrifuge fractionation after dispersion of the total soil 
by agitation with glass balls and dispersion of the fraction <50 jam with 
ultrasounds (Balesdent et al.1991) 

The above conditions were calculated by Balesdent et al. (1991) for 
Stokes diameters of 0.2 or 2 jim, a particle density of 2.5 g cm -3 and the 
data specific to their equipment. The density used corresponds more to 
mineral than to organic particles. Thus the fractions indicated do not 
strictly correspond to the size of organic particles, but it is difficult to 
separate inorganic and organic matters that are associated in the fractions 
of clay size. After each decantation of the 0-0.2 or 0.2-2 |im supernatant, 
suspend the centrifugation pellet in water, agitate for 30 min and 
centrifuge again. These authors advised four sedimentations at 0-0.2 |im 
then four at 0.2-2 |im. Centrifuge the 0.2-2 |im suspension for 25 min at 
2,900g and recover the centrifugation pellet. Mix the supernatant with the 



318 Organic Analysis 

previously obtained 0-0.2 |um fractions. Flocculate the suspension by 
adding a 0.5 g (CaCl 2 ) L _1 solution; store overnight and centrifuge. The 
centrifugation pellet is the 0-0.2 |um fraction and the supernatant is the 
final organic water-soluble fraction. 

Fractionation of the clay size particles can be performed more easily 
by continuous flow ultra-centrifugation (cf. Chap. 2). 

The fractions over 50 |um should be dried at 60°C, those below 50 |um 
should be homogenized, frozen and freeze-dried. They are weighed and 
then crushed to 50 |um for chemical analyses, especially measurement of 
their C and N content (cf. Chap. 10). 

Fractionation using Resin H + and ultrasounds 

Decantation method. In this procedure described by Feller et al. 
(1991), the initial dispersion of the soil is carried out with a cation exchange 
resin (Amberlite IRN77 in Na + form) carefully sieved to 500 |um. The 
sieving operation must be renewed before each fractionation. Split the 
resin 100-mL portions and place them in polyamide bags (Nytrel TI45) 
with a mesh size of 45 |um. Place these bags in 60-|um mesh bags (Nytrel 
TI60). Close the bags with a rubber band. The double bag protects the 
soil against contamination in the event the bag should break. 

Dispersion is then carried out by agitation of 20 g air dried soil for 16 
h with 300 mL distilled water in a 1 L bottle containing one resin bag. 
Remove the bag from the suspension, wash abundantly with water and 
reserve for measurement of the small quantities of 20-50 |um soil fraction 
that may be trapped in the bag (weigh the fraction remaining on a 20-|um 
sieve after recovery and wash the resin on a 50-|um sieve). 

The remaining operations can be performed following the procedure 
described in "Agitation with Glass Balls and Ultrasonification". However, 
the procedure of Feller et al. although very similar to the previous 
section, includes slight differences in ultrasonic energy, and in the clay 
fractionation method. Sieve the soil-water suspension to 200 and 50 |um. 
Wash the material remaining on the mesh of the sieves and subject the 
0-50 |um suspension obtained in fractions of 1 L to ultrasounds. The 
apparatus (250 TH, US Annemasse) uses a frequency of 20 kHz, variable 
electric output (0 to 300 W), and is equipped with a probe with a flat head 
9 mm diameter. This sounding head is located 2.5 cm from the bottom of 
the suspension, ultrasound is applied continuously for 7 min at 75% of 
maximum capacity, i.e. 0.23 W mL -1 suspension, approximately 100 J 
mL- 1 . 

Sieve the 0-50 |um suspension, wash the material remaining on the 
mesh, then transfer the 0-20 \im suspension in two sedimentation 
cylinders and bring to 1 L with distilled water. Shake the cylinders by 



Physical Fractionation of Organic Matter 319 

turning them upside down and back (30 reversals) and place them on the 
lab table during sedimentation of the 0-2 |um fraction (cf. Chap. 2) for 
subsequent pipette sampling of this fraction. Repeat this operation until 
exhaustion (minimum five times). The sediment remaining at the bottom 
of the cylinder is the 2-20 |um fraction. Centrifuge the sampled 0-2 |um 
suspensions for 1 h at 2,500g to separate the centrifugation pellet (0.2- 
2 |um) and the 0-0.2 |um supernatant. Repeat this operation twice. 
Flocculate all the collected supernatants by additions of 2 mL L _1 of 
saturated SrCl 2 . Separate the clear supernatant from the centrifugation 
pellet (0-0.2 |um fraction) by centrifugation. 
The following fractions are obtained: 

- by wet sieving, the 200-2000, 50-200 and 20-50 jim fractions 

- by sedimentation, the 2-20, 0.2-2 and 0-0.2 |um fractions 

- a water-soluble organic fraction. 

Depending on the type of soil, or when too energetic dispersion is not 
desired, the same technique can be used on the 0-50 |um suspension 
without the ultrasound treatment. 

Method with sampling of aliquots. This method was described by 
Gavinelli et al. (1995) and is faster than the preceding one for measurement 
of the silt and clay fractions. Using a Robinson pipette, remove aliquots 
from sedimentation cylinders (cf. Chap. 2). 

Special Procedure for Sandy Soils 

The procedure of Feller (1979) was developed on sandy soils. It includes 
low energy mechanical dispersion by agitation of the soil-water 
suspensions (100 g soil-300 mL water) for 1 h with three glass balls. 
Sieving with water followed by separation of the fractions as in "Resin 
H + and Ultrasounds" enables recovery of the M2000, F2000, M200, 
F200, M50, F50, OM, W fractions (M: organomineral fraction, F: organic 
fragments, number: lower limit of particle size of the fraction, OM: 
organomineral fraction below 50 |um separated by centrifugation, W: 
water-soluble fraction). 

The procedure described in "Resin H + and Ultrasounds" also includes 
one modification for use with sandy to clayey-sandy soils. The length of 
agitation of the soil-water-resin suspensions in the bags is reduced to 
avoid too much deterioration of the plant debris by sands. The procedure 
is as follows: 

Place 40 g of air-dried soil in a 1 L bottle with 300 mL distilled water 
and a 100 mL bag containing "Amberlite IRN77" cation exchange resin 
in Na + form (cf. "Resin H + and Ultrasounds"). Agitate the bottles on a 
back and forth shaker for 2 h at moderate speed. Separate the fractions 



320 



Organic Analysis 



above 50 |um by sieving. Agitate the 0-50 urn suspension for 14 h with 
the resin. The remaining operations are identical to the procedure 
described in "Resin H + and Ultrasounds" of this chapter. 

9.2.6 Precision of the Fractionation Methods 

The precision of the techniques for physical fractionation of organic 
matters varies considerably with the type of soil and especially with the 
stage of development of the organic matter. In general, the smaller the 
quantity of the fraction, the greater the variability. Repeatability increases 
with the particle size of the fraction. Relative error resulting from 
fractionation varies in the same way for percentages by weight or the 
percentage of the carbon of the fraction compared to total carbon. 

Because of its weak relative weight, the error on the determination of 
the coarse and light organic fraction is often the most significant. This 
error also appears to be linked to the method since Monnier et al. (1962) 
found for four types of soil, variations ranging from +30 to +60% when 
comparing the funnel method of Jeanson-Luusinang (1960) with the 
centrifugation method. Oudinot (1985) found for the fraction with a 
density lower than 1.4, a relative standard deviation of 28% in the case of 
a calcareous brown soil and 62% in the case of a fersiallitic soil. Feller 
(1979) also found a coefficient of variation of 63% calculated on 60 
replicates of measurements of a coarse organic F2000 fraction separated 
by sieving at 2 mm and floating in water. Monnier et al. (1962) obtained 
for two replicates on four types of soil a pooled relative standard 
deviation of about 2%. 

Table 9.1. Error in the precision of the particle size fractionation of a sandy soil 
from Senegal (Feller 1979; F: organic fragments, M: organomineral 
fraction, number: lower particle size threshold, OM: organomineral 
fraction <50 |im, W: water soluble organic fraction), m: carbon percent 
of the carbon of the sum of the fractions, RSD: relative standard 
deviation in percent of measurement for ten replicates 



fraction 


m 


RSD(%) 


F2000 


3.0 


63 


F200 


21.6 


11 


M200 


1.2 


42 


F50 


8.8 


25 


M50 


5.8 


28 


OM 


57.6 


6 


W 


2.0 


25 



Physical Fractionation of Organic Matter 



321 



Table 9.2. Repeatability of the particle size fractionation of a ferrallitic soil and a 
vertic soil (Feller et al. 1991). RSD% m and RSD% Ct = relative 
standard deviation of the mass fraction and the carbon fraction (total 
carbon of fraction/total carbon) for four replicates 



soil type 


fraction (jim) 


RSD% m 


RSD% Ct 


Ferrallitic 


0-2 


10 


23 




2-20 


25 


48 




20-2000 


8 


48 


Vertic 


0-2 


4 


8 




2-20 


4 


14 




20-2000 


10 


30 



Feller (1979) also measured the error in the percentages of carbon for 
each fraction (compared with total soil) obtained by wet sieving and 
decantation (cf Special procedure for sandy soils). The results (Table 9.1) 
underline the significance of the error in precision with respect to quanti- 
tative studies of soil organic matter and how error varies with the size of 
the fraction. 

The study of Feller et al. (1991) also included an evaluation of 
precision related both to the mass of the fractions and their carbon 
contents compared to total soil carbon (Table 9.2). The error was shown 
to depend on the type of soil. The error was lower in the case of a vertic 
soil than in a ferrallitic soil. 



9.3. Conclusion and Outlook 



Physical fractionation techniques are often used before other studies on 
soil organic matter. Indeed, they themselves comprise one of the methods 
of the study of organic matter. 

No method enables perfect separation of each component of the soil 
(plant roots, plant fragments, animal fragments, micro-organisms and 
metabolites of organomineral complexes). The methods described in this 
chapter seem to be the most suitable for further development. They have 
been classified under three main functions: extraction of plant roots; 
extraction of "free organic matter" corresponding to organic fragments 
that have not completed deteriorated; fractionation of organic matter in 
particle-size ranges. 



322 Organic Analysis 

Apparatuses for root-soil separation are based on the principles of 
elutriation and underwater sieving. Their complexity and the fact that the 
operations of separation they perform are not exhaustive, led some 
authors to prefer manual techniques. 

Density techniques are relatively simple to use. Coupling density with 
particle size fractionation of the coarse particles and the use of not very 
aggressive methods of dispersion of the structural aggregates increases 
reliability. 

The techniques of particle size fractionation enable more extended 
classification of organic matter, in particular of the three main organic 
and organomineral compartments mentioned by Feller (1994). 

Along with a description of the techniques of physical fractionation, 
this chapter describes the main types of soil on which the techniques were 
tested. Adaptations are probably necessary for other soils or to fulfil 
certain specific research objectives. These adaptations should be also 
helped by observations of Christensen (2001), Six et al. (2002), Rovira 
and Vallejo (2003) or Xu et al. (2003). The observations in this chapter 
should be taken into account, especially precautions related to the use of 
ultrasounds and dispersing agents; the aim being to obtain better 
separation of organic fragments and organomineral complexes with less 
destruction of organic entities. 



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10 



Organic and Total C, N (H, O, S) Analysis 



10.1 Introduction 



10.1.1 Soil Organic Matter 

Organic matter plays a determining role in pedogenesis and can 
drastically modify the physical, chemical, and biological properties of 
soil (structure, plasticity, colour, water retention, CEC, and AEC). 

The fundamental processes of evolution include phenomena of 
mineralization and immobilization and, in particular, of carbon and 
nitrogen. Mineralization allows the transformation of organic residues 
into inorganic compounds in the soil, the atmosphere, and the hydrosphere, 
these are then usable by flora and by micro-organisms. Immobilization is 
the transformation of organic matter into more stable organic and organo- 
mineral compounds with high molecular weights that are fixed in the 
interlay er spaces of clays. These processes are summarized by the 
following diagram: 

Soil organic Mineralization CQ ^ CH ^ 

matter and 

necromass Immobilisation 



NH 3 t ^NH 4 ^ 

Tn 2 o, n 2 , no x 



Gams Losses N0 2 ^N0 3 

Apparent 

equilibrium 



328 Organic Analysis 

This cycle includes phases of mineralization, humification, 
ammonification, immobilization, nitrification, and volatilization under the 
action of specific micro-organisms (Pansu et al. 1998) and is influenced 
by a number of factors, of which the most significant are: 

- climate (temperature and moisture and their effects on microbial 
activity, micro-fauna and micro-flora) 

- topography 

- types of vegetation and litters 

- the nature of the parental material (mainly texture, mineralogy and pH) 

- time (age of the soil and state of equilibrium) 
and, in addition, for cultivated soils 

- the effects of farming practices such as ploughing, irrigation, burning, 
addition of manure, fertilizer, pesticides 

- types of crops, exports by crops and use of crop residues. 

Soil C and N contents can vary considerably, e.g. a tropical oxisol can 
contain less than 2% of total C while in andosols and histisols, total C can 
exceed 30%. 

The dynamics of the transformation of C and N are very complex and 
difficult to model accurately. In addition to the measurement of total C 
and N, a simple index is needed to clarify the dynamics and allow 
samples from different climatic zones to be compared, as well as to 
identify the evolution of the organic matter in a soil profile, or the 
presence of a buried organic A horizon. 

The first in situ observations of humified litters (A o) of the organic A 
horizon (which are of varying thickness) and of the eluvial or illuvial 
horizons provide very important information. The evolution of the 
organic matter is then analysed by studying its chemical structure and 
physicochemical properties. The different forms of matter can be 
separated into characteristic entities of varying degrees of polymerization 
and quantified by physical separation (cf. Chap. 9) by their resistance to 
hydrolysis (in acid and basic media) and their solubility in specific 
solvents and reagents (cf Chaps. 11 and 12). Laboratory techniques such 
as gel permeation chromatography allow determination of the molecular 
weights of the humified substances after purification. UV, visible, or IR 
absorption and other spectrographic methods allow identification of the 
molecular structures and of rates of polymerization. Both active 
functional groups and the formation of the clay-humus complexes can be 
studied (cf. Chap. 12). The elementary analysis described in this chapter 
enables the total chemical composition of the organic matters to be 
established. 

The different factors that control humification, especially climate, 
parental material, and biomass, result in physicochemical constraints that, 



C, N (H, O, S) 329 

in turn, result in a given type of humus e.g. Mor, Moder, forest and calcic 
Mulls, Anmoor or peats. Microscopic observation (cf. Chap. 8) of the 
morphology of the systems at different scales allows characterization of 
the interfaces of organic and inorganic matter and of the mechanisms of 
humification. Kinetic methods can also be used to analyse the 
biogeochemical dynamics of the organic residues: 

- By measuring, for example, the C0 2 released per unit of time by means 
of portable chromatographs or IR captors installed in situ (respirometry, 
biological evolution). 

- By using 14 C, 13 C, and 15 N isotopic tracers to monitor the transformation of 
organic substances added to the soil (turnover rate of soil organic 
matter). 

- By studying the differentiation of stable isotopes such as 13 C, 14 C, or 15 N and 
and isotopic ratios for studies of paleoclimatology and geochronology. 

Table 10.1. Typical C:N ratios of a few main types of humus 



type 


C:N 


pH range 


calcic eutrophic Mull 


= 10 


7.0-8.4 


forest Mull 


12-15 


5.5-6.5 


moder 


15-25 


4.0-5.0 


mor 


>20 


<4.0 




calcic eutrophic peats 


<30 


- 


hydromorphy 


acid oligotrophic peats 


= 40 


- 




anmoor 


variable 


- 



The majority of these methods require a high degree of specificity and 
highly sensitive sensors because of the scales of measurement needed to 
measure extremely weak variations. They are time consuming and 
expensive and are not sufficiently universal for use in serial analysis. On 
the other hand, total-C and -N can be measured using simple methods that 
are accessible to all laboratories. Improvement in equipment for dry 
analysis (e.g. CHN(OS)) now makes it possible to standardize analyses 
and combine precision, speed, and automation. Some of this equipment 
can handle representative samples of more than 100 mg. 

The C:N ratio of the soil in the surface horizons can be determined 
only using information on total C and N (Table 10.1). This information 
can then be used as an index that provides relatively reliable information 
on the biological activity and equilibrium of the two elements that have 
been subjected to the antagonistic processes of mineralization and 
immobilization. In regions with a temperate climate, the C:N ratio is 



330 Organic Analysis 

about 10-12 for uncultivated soils and generally decreases with an 
increase in soil depth. In certain soils N can be significantly occluded in 
clays, especially in deep horizons. In forest soils, peat horizons, or 
podzols, C:N ratios can reach 20-30 or even higher because of the 
formation of only slightly biodegradable complexes which are low in 
nitrogen (e.g. Spodic horizons). At C:N ratios below a threshold near 20, 
positive N net mineralization is generally observed. In cultivated soils, 
farming residues recycled in the field have C:N ratios ranging between 15 
and 60 due to the presence of lignin-cellulose compounds with a slow 
rate of degradation. Under forest with acidifying litter, the C:N ratios can 
reach 150 or even higher. 

10.1.2 Sampling, Preparation of the Samples, Analytical 
Significance 



Equipment 

- strainer with 2 mm round holes, AFNOR NF34. 

-cutting grinder equipped with a 125 |Lim mesh sieve (AFNOR NF22) 
and a watertight collector (for litter). 

- grinder with retractable hammer equipped with an AFNOR NF22 sieve 
and a watertight collector (mineral or organo-mineral horizons). 

- agate mortar and pestle. 

-analytical balances (±0.1 mg or ±0.01 mg depending on test 
specimens). 

- drying oven regulated at 105°C. 

Procedures and Precautions 

As the heterogeneity of the soil surface horizons near the litters is very 
high, sampling is difficult and must be carried out with great care. The 
way samples are collected will subsequently affect the validity of the 
results. Drying should be carried out in contact with air in a well- 
ventilated room. 

At the laboratory, samples should be crushed to 2 mm to separate non- 
decayed or only slightly decayed plant debris. Care should be taken to 
avoid breaking up the organic fragments that have retained their original 
texture as this could overload the sample significantly. This stage affects 
the significance of the analytical result as does the weight of the test 
specimens during analysis (Pansu et al. 2001). 



C, N(H,0, S) 331 

Drying increases the fixing of ammonium particularly if the parental 
matrix contains 2:1 clays such as montmorillonite or vermiculites. The 
action of the micro-organisms may not have stopped during storage 
depending on soil respiration, lignin content, or on residual moisture in 
the air-dried sample (in andosols and histisols, the moisture rate of air- 
dried soil can still be 60% after 6 months). 

Grinding a whole 2-mm particle-size sub-sample to 125 |Lim particle 
size (AFNOR NF22 sieve) can modify the moisture and equilibrium of 
the reactive surfaces. Moisture content is used to correct the analytical 
results and must be measured at the same time as analytical sampling is 
performed. If drying at 105°C does not exceed 3 or 4 h, there is generally 
no significant loss of C and N in gaseous form; but drying can slightly 
increase fixing of N in the clay lattice. In the case of instrumental 
analysis (CHN(OS)), drying the samples at 105°C can avoid the need to 
correct the results and can limit clogging of the water traps. 

Careful grinding into fine particles that are homogenous in size is 
necessary to improve the reproducibility of the results, soil powder 
sampling can vary between 5 and 500 mg depending on the instrument 
used for dry analyses (CHN(OS)). In the case of wet analyses, grinding 
ensures a more regular and complete attack by considerably increasing 
the solid-liquid interfaces. 

Expression of the Results 

If the analytical results are to be used for agronomic purposes, it is 
advisable to take the soil density into account, particularly in the case of 
peats, andosols, and histisols where apparent density can approach 0.30. 
In this case, the concentration expressed per mass unit may be far from 
the inorganic contents actually available for the plants per unit volume 
and it is thus essential to correct for density. 

In the case of soils with high contents of gravels, stones, rocks, or non- 
decayed plant debris that display a significant rejection rate during 
preparation of 2 mm soil samples, it is also better to correct the rough 
results of analysis to obtain a value approaching the quality of the soil 
per unit of volume. 

Preliminary Tests 

A good knowledge of the formation processes and agricultural use of the 
soils concerned makes it possible to limit the number of tests required. 



332 



Organic Analysis 



The number of tests also depends on the analytical method to be used 
(Table 10.2). 

It should be noted that by convention, the "total organic matter of the 
soil" corresponds to the transformed organic forms and excludes intact 
plant and animal residues. In practice, since the separation of light or 
non-decayed fragments of organic matter (cf. Chap. 9) is difficult in 
repetitive analysis, particles of a size lower than 2 mm are considered to 
be an essential part of the sample. Living micro-organisms are integrated. 
A range of different types of tests can be carried out to obtain more 
detailed knowledge on the analytical substrate to be in a position to 
choose the appropriate analytical procedures. 

Examination with a magnifying glass enables confirmation of presence 
of seashells, limestone amendments, coals, etc. (soils under crops, coastal 
soils, calcareous soils, etc.). 

HCl Tests: below pH 7.4-7.0, indicates the presence of carbonates and 
bicarbonates only in the form of isolated particles. 

Table 10.2. Analytical methods 



method 



source of interference 



type 



dry 
methods 



combustion < 360°C 
combustion > 360°C 



gypsum, losses of water above 150°C 
various forms of water (hydration) 
carbonate decomposition C0 2 t 
Na 2 C0 3 melting (clogging) 
various forms of water (bound water) 



wet 
methods 



classical N Kjeldahl 
method 



cold C oxidation 



hot C oxidation 



N0 3 ~, N0 2 " random recovery (not 
quantified if not previously reduced) 
NH 4 + fixed in the crystal lattice of 2:1 
clays (random recovery) 



results are too low (multiplicative factor) 
presence of chloride, oxidative and 
reducing agents 



presence of chloride, oxidative and 
reducing agents 



N0 3 ~, N0 2 ~ test: a rapid test using a soil analytical kit is useful in 
intensively cultivated and waterlogged soils. 

Fixed NH 4 test: only used in soils containing 2:1 clays to check 
ammonium concentration with two different methods and possibly by 
destruction of the crystal lattice (cf. Sect. 28.3.5 of Chap. 28). 



C, N (H, O, S) 333 

Preliminary Destruction of Carbonates and Bicarbonates (Dry 
Combustion Methods) 

The samples should be treated at room temperature with a 0.1 mol (HO) 
L" 1 solution until the end of the reaction. If the presence of dolomite, 
siderite, or biogenic calcite is suspected, contact time should be increased 
to 2 or 3 h. 

CaC0 3 + 2H + -> Ca 2+ + C0 2 T + H 2 
Samples containing siderite can be treated using the hot H 3 P0 4 or 
CH3COOH 0.3 mol (H + )L _1 for 5 h. Destruction is difficult and often 
incomplete. Dry carefully. 

Total C - inorganic C = organic C (10-1) 

Care should be taken to not lose soluble organic C in the acids (e.g. 
from amino-acids or phospholipids). Formation of hygroscopic salt can 
disturb weighing. The presence of manganese dioxide in the soil can 
cause the release of Cl 2 starting from HC1. 

In arid or semi-arid regions, soluble salts that may be present (e.g. 
carbonates, chlorides, or sulphur compounds) can slow down the organic 
oxidation of C because of their low melting points (e.g. sodium 
carbonate) and consequently disturb measurements by dry combustion. 



10.2 Wet Methods 



10.2.1 Total Carbon: General Information 

Strictly speaking the "total carbon" of the soil comes from two principal 

sources (10.1): 

- Organic carbon (only slightly processed organic residues of plant and 
animal origin, humus, charcoal, fossil organic matter, micro- 
organisms). 

-Inorganic carbon possibly present in the form of carbonates and 
bicarbonates. 
In the majority of methods, the gas phases present in the atmosphere of 

the soil (C0 2 linked with biological activity, CH 4 ) are not taken into 

account. 

Some ambiguity persists in the terminology and methods used. 

Measurements carried out on non-processed soil samples (without 

preliminary elimination of carbonates) using dry combustion methods in 



334 Organic Analysis 

CHN(OS) apparatuses give organic and inorganic forms of "total 
carbon". Measurements by oxydo-reduction using wet oxidation give 
only "organic carbon" corresponding to humified forms and organic 
matter of debris that are still rich in non-transformed cellulose, but does 
not include charcoal or fossil organic matter. Although wet methods at 
room temperature do not allow complete attack of the humus (a correction 
index will be needed), they are nevertheless used for the determination of 
"total organic carbon". 

"Total inorganic carbon" can be measured using the methods described 
in Chap. 17 but with varying precision due to the slow chemical 
decomposition of magnesium carbonate (MgC0 3 ) and especially of 
siderite (FeC0 3 ). 

The term "total organic matter" is often used. Empirically it expresses 
"total organic carbon" determined by oxidation-reduction but corrected 
by a coefficient based on the assumption that organic matter contains 
mainly humic acids at approximately 58% of carbon (100/58 = 1.724 van 
Bemmelen factor). In fact this rate is far from constant even for the 
horizons near the soil surface. The coefficient is thus not very realistic, 
particularly for soils containing not very humified Mor or Moder, forest 
soils, and peats. In these cases a coefficient of up to 2 or even 2.5 will be 
required. The term "total organic matter" is thus an estimation and cannot 
be used as an index. 

In practice, the term "total carbon" is incorrectly used by some authors 
to indicate "total organic carbon" as well as to establish balances in total 
analysis, C:N ratios, or to compare the total C contents of the different 
horizons of a soil profile and the organic distribution of C as a function of 
depth. 

The term total organic carbon should cover all the organic substances 
resulting from the humification of C in the soil (microbial residues, 
humic substances) under the influence of biochemical and chemical 
reactions. Additionally it represents light organic matter still not 
completely decomposed that could not be separated on a sieve during the 
preparation of the samples (litters, coarse organic plant or animal 
fragments under 2 mm in size). Fossil organic matter (coal, naphthas, 
resins, etc.) and charcoals in regions exposed to forest fires or in regions 
with slash and burn agriculture are not subject to this type of dynamics, 
which means they do not have to be taken into account when wet redox 
methods are used. However they are always included when dry 
combustion methods are used and this can lead to difficulties when the 
results obtained by the two methods have to be compared. 

Thus the study of the total organic carbon stock may need to be 
refined: 



C, N (H, O, S) 335 

- By micro-morphologic observations at different scales to determine the 

relative proportions of the contents of unprocessed and humified 

organic matter together with selective extractions in different mediums. 

-By the determination of the origin, the nature and the rates of 

mineralization of the different forms as a function of the pH, the nature 

of the clays and clay-humus complex, and of soil management practices. 

Wet methods require only relatively inexpensive equipment, which 

means they can be used in all laboratories. These methods make it 

possible to work with big samples which are more representative of the 

natural environment. On the other hand, they are time consuming, require 

the handling of very corrosive products and the elimination of polluting 

products (Cr 3+ , H 2 S0 4 ), which can pose problems for the environment. 

10.2.2 Organic Carbon by Wet Oxidation at the Temperature 
of Reaction 



Introduction 

The determination of total organic carbon by oxidation with potassium 
dichromate in a strong acid open medium, was proposed first by 
Schollenberger (1927) then by Walkley and Black (1934) from which 
it takes its name. After a stage of oxidation/mineralization at the temperature 
of reaction for a given length of time, the non-reduced dichromate in 
excess is back titrated by ferrous iron. 

Many authors have studied the factors that affect C mineralization: 
acid concentration (H 2 S0 4 , H 3 P0 4 ), potassium dichromate concentration, 
oxidation temperature (from the temperature of reaction to +210°C), the 
time of contact, the need to condense the vapour to avoid too high 
concentration of the medium, and to limit destruction of the oxidant by 
avoiding overheating of the walls. The choice of the temperature resulted 
in two different types of methods: 

- At the temperature of reaction = 120°C (Walkley and Black 1934). 

- At standardized boiling = 150°C (Anne 1945; Mebius 1960). 

Different procedures were proposed for back titration of dichromate 
excess such as soil/solution separation by centrifugation and filtration, 
but direct volumetric titration in the soil suspension was the most widely 
adopted. Redox indicators (diphenylamine, barium diphenylamine 
sulphonate, N-phenyl anthranilic acid, ferrous O-phenanthroline) can be 
absorbed on clays. Additives (e.g. NaF, H 3 P0 4 ) allow better reading by 
sequestering the coloured products that are formed or dissolved (e.g. 
Fe 3+ ) and which can mask the reaction. 



336 Organic Analysis 

Principle 

Mineralization 

Organic forms of C are oxidized in the presence of excess dichromate. 
The reaction in a concentrated acid medium is exothermic (= 120°C). It 
develops at a fast kinetics under the following conditions: 

3C + 2Cr 2 7 2 " + 16H + 12 °° C > 4Cr 3+ + 8H 2 + 3C0 2 
The amount of reduced dichromate is considered to be quantitatively 
linked to the organic C content of the sample. The likelihood of reduction 
is assumed to be identical for different forms of organic C and the 
reducing power is assumed to be constant during mineralization. In 
practice, at the temperature of reaction without heating, a factor of 
correction will be required because only the most active forms are 
oxidized, i.e. 60%-80% of organic matter. This factor was fixed at 1.30 
(100/76) to take the variable reactivity of the organic forms into account, 
but can vary between 1.10 and 1.45 depending on the soils and on the 
types of vegetation. 

The inorganic forms (carbonates, bicarbonates) are destroyed and do 
not play any role except in the consumption of acid and the production of 
foam. The precipitation of calcium sulphate can be problematic during 
final titration if a spectro-colorimetric method is used. 

CaC0 3 + H 2 S0 4 -> CaS0 4 i + H 2 + C0 2 T 

Titration 

Volumetric back titration of the Cr +VI dichromate not consumed by 
organic C is carried out by reduction (ferrous sulphate or Mohr's salt) in 
the presence of an indicator. 

Cr 2 7 2 " + 6Fe 2+ + 14H + -> 2Cr 3+ + 6Fe 3+ + 7H 2 
Sodium fluoride or phosphoric acid (H 3 P0 4 ) can be added to fix the 
ferric iron that is formed or dissolved, and to improve the detection of the 
equilibrium point of titration: 

Fe 2 3 + 3H 2 S0 4 -> Fe 2 (S0 4 ) 3 + 3H 2 
Fe 3+ + 6F" — > FeF 6 3 " (uncoloured) 
Nevertheless, the addition of fluoride can result in the formation of 
hydrofluoric acid which attacks glass and silicates: 

2NaF + H 2 S0 4 -> Na 2 S0 4 + 2HF 
Si0 2 + 6HF -> H 2 SiF 6 + 2H 2 



C, N (H, O, S) 337 

It is thus necessary to clean the lab glassware immediately after 
titration and to reserve this glassware for the determination of C using 
this redox method. 

Interferences 

In saline soils, chlorides cause a positive error by forming chromyl 
chloride: 

K 2 Cr 2 7 + 6H2SO4 + 4KC1 -> 2Cr0 2 Cl 2 + 6KHSO4 + 3H 2 

Ferrous iron that may be present is oxidized by dichromate and thus 
modifies the quantity of ferrous sulphate necessary for back titration. 
Corrections will be necessary in waterlogged soils in which Fe 2+ is 
sometimes abundant. 

The method cannot be used in acid sulphated soils that are rich in 
pyrite (FeS 2 ): 

5Cr +VI + FeS 2 -> Fe 3+ + 2S +VI + 5Cr 3+ 

A high level of Mn 2+ can also interfere, as can iron metal that can 
results from wear of the grinding equipment. 

Equipment 

-Analytical balance (±1/10 mg) and a top-loading balance with a 
capacity of 500 g (±1 mg). 

- 500 mL wide-neck Pyrex Erlenmeyer flasks. 

- Insulating plates. 

- Teflon flask dispenser. 

- Burette for titration. 

- Magnetic stirrer with Teflon bars. 

Reagents 

All the reagents should be of analytical reference grade: 

- Distilled or bi-distilled water (avoid water that has been deionised by 
ion exchange as it can contain fine particles of ion-exchange resins). 

- 1 mol (e) L" 1 potassium dichromate (standard): in a 1 L volumetric 
flask, dissolve 49.040 g of K 2 Cr 2 7 (dried under vacuum or on P 2 5 in 
a desiccator) in 800 mL of bi-distilled water, then adjust to 1,000 mL. 

- Concentrated sulphuric acid, H 2 S0 4 d= 1.84. 

- 0.5 mol (H + ) L" 1 sulphuric acid solution: in a 1,000 mL Pyrex 
volumetric cylinder, add 800 mL distilled water, then slowly add 13.9 
mL of concentrated sulphuric acid, homogenize. Allow to cool and 
bring to 1 L with distilled water. 



338 Organic Analysis 

- 0.5 mol (e) L" 1 iron and ammonium sulphate (Mohr's salt): in a 1,000 
mL volumetric flask, dissolve 196.05 g of Fe(NH4) 2 S04,6H 2 (dried on 
P 2 5 in a desiccator) in approximately 800 mL of 0.5 mol (H + ) L" 1 
H 2 S0 4 solution. Adjust to 1,000 mL with the 0.5 mol (H + ) L" 1 H 2 S0 4 
solution. The liquid should be clear and pale green in colour. 

- Concentrated phosphoric acid H3PO4 d= 1.71 (85%). 

- Sodium fluoride NaF in powder form. 

- Diphenylamine V^ \_J^ solution: dissolve 0.5 g diphenylamine 
in 100 mL of concentrated H 2 S0 4 . Pour into 20 mL water and store in a 
brown glass bottle with a ground stopper with dropping pipette. 

Other indicators can be used: 




o-Phenanthroline (1-10 phenanthroline) \^\^ which forms a 
ferroine complex with Fe 2+ : dissolve 14.85 g o-phenanthroline 
monohydrate and 6.95 g ferrous sulphate (FeS0 4 , 7H 2 0) in 800 mL 
distilled water. Bring to 1 ,000 mL in a volumetric flask and store in a 
brown glass bottle. 

Barium diphenylamine sulphonate Ba(C 6 H 5 -NH-C 6 H4-S03) 2 . Dissolve 
in distilled water. 

COOH 




- N phenyl anthranilic acid 

Procedure 

If total N was analysed beforehand, determine the approximate weight of 
soil required to obtain a sample specimen containing between 10 and 25 
mgC (Table 10.3). 

Weigh this sample specimen (± 0.1 mg), transfer it in a wide-neck 500 
mL Erlenmeyer flask and add exactly 10 mL of the potassium dichromate 
1 mol (e) L" 1 solution. Homogenize carefully to avoid making the 
suspension go up the walls of the flask. Quickly add 20 mL of 
concentrated sulphuric acid with a Teflon dispenser. Agitate by rotation 
for one minute to homogenize (the temperature of reaction is approximately 
120°C). Place on an insulating plate and let oxidation take continue for 
30 min. 

Add 200 mL distilled water, then 10 mL of phosphoric acid (or 
approximately 5 g of sodium fluoride with a suitable spatula). 
Homogenize. Add three drops of diphenylamine. 



C, N(H,0, S) 



339 



Titrate the excess dichromate with the 0.5 mol (e) L" 1 ferrous iron 
solution (this reagent should be freshly titrated each day). The end of 
titration is indicated by the change in colour from purplish blue to a 
rather luminous greenish blue. Determination of the end point is 
facilitated by adding 1-2 drops of indicator as soon as the colour begins 
to change. 

Note: the solution should still be orange after the attack of the organic matter 
indicating an excess of dichromate. If the solution is green, start again 
using a smaller sample of soil. 

Table 10.3. Recommended size of test specimen (P g of soil sample) as a 
function of nitrogen content (on the basis of a C:N ratio of 10). 



Ng kg 



sample 



mg C sample 
(theoretical) 



Ng kg 



sample 
PQ 



mgC 

sample 

(theoretical) 



0.1-0.2 
0.3-0.4 
0.5-0.9 



10 
5 
2.5 



10-20 
15-20 
12.5-22.5 



1-2 
3-4 
5-10 



1 

0.5 

0.2 



10-20 
15-20 
10-20 



Expression of the Result 

It is an accepted fact that the oxygen consumed is proportional to the 
carbon titrated on the theoretical basis of 1 mL of 1 mol (e) L" 1 
dichromate solution oxidizing 3 mg C, i.e. corrected by the attack 
coefficient (1.3 = 100/76) = 3.9 mg C. This attack coefficient can be 
modulated as a function of the form of C and by comparison with 
measurements made by dry combustion: 
-Total organic C gkg 1 of 105°C dried soil = 3.9(10-0.5 V)j 

where P is the sample mass in g, V is the volume (mL) of Fe 2+ solution 
at a concentration of 0.5 mol L" 1 (replace 0.5 by the exact concentration 
if it is not exactly 0.5) and the quantity of dichromate solution added is 
10 mL. 
- Total organic matter g kg" 1 = total organic C x 1.724 

Control 



Each day, make two measurements with 10 mL 1 mol (e ) L 
solution to check the exact concentration of the Fe 2+ solution 



-l 



dichromate 



340 Organic Analysis 

In each series, carry out two blank titrations with quartz prefired at 
1,100°C, and two titration controls on samples of a reference soil of the 
same type as the soils being analyzed. 

10.2.3 Organic Carbon by Wet Oxidation at Controlled 
Temperature 



Introduction and Principle 

When measurements are made using wet oxidation at the temperature of 
reaction, the mineralization/oxidation of active organic carbon is always 
incomplete. The use of a "standardized" corrective factor of 1.3 introduces a 
variable that is not easily controllable because in practice, this coefficient 
ranges from 1 . 1 to 1 .40. 

To mitigate this problem, Anne (1945) then Mebius (1960) proposed 
carrying out total mineralization by maintaining the sample at a constant 
temperature throughout the process of mineralization without causing 
thermal decomposition of the dichromate. Effectiveness and reproducibility 
were tested between 130 and 210°C with different times of oxidation and 
different acid/dichromate ratios. Above 150°C the dichromate tends to 
decompose more and more quickly, thus necessitating relatively short 
attack times. Strict respect of the procedure and precise control of the 
attack times and the temperature enable an acceptable level of accuracy. 
In principles this type of measurement and possible interferences are the 
same as for the method described in Sect. 10.2.2 earlier. 

Equipment 

-A mineralization block (with from 20 to 40 places regulated 
thermostatically at 150°C (Tecator, Skalar, Technicon, etc.); mechani- 
zation is possible in laboratories that carry out many repetitive 
analyses; in the Skalar system, for example, a sample holder is capped 
by a device with 20 reflux condensers. After introducing the samples 
and reagents, place the unit in the programmed heating block; after the 
period of mineralization, remove the unit and cool, separate the rack 
condensers; the sample holder advances on rails towards the dilution 
stage and possibly towards the titration system. 

- Analytical balance (±1/10 mg). 

- Digestion tubes with ground joint for the condenser. 

- Titration burette. 



C, N (H, O, S) 341 

-Precision volume dispenser (±1/10 mL) with Pyrex glass syringe and 
Teflon piston. 

- Magnetic stirrer with 15 mm Teflon bars. 

Reagents 

- cf. "Reagents" under Sect. 10.2.2. 

- 0.2 mol (Fe 2+ ) L" 1 solution: weigh 78.5 g of Mohr salt (dried in P 2 5 
desiccator), dissolve in 800 mL of 0.5 mol (H + ) L 1 sulphuric acid 
solution. Bring to 1 ,000 mL with the sulphuric acid solution. 

Procedure 

Weigh (±0.1 mg) between 200 mg and 2 g of soil (ground to 125 |Lim 
particle size and dried on P20 5 ) to have a sample containing 
approximately 15 mg C (Table 10.3). 

In a Pyrex tube (with ground joint for the condenser), add 10 mL of 1 
mol (e) L" 1 potassium dichromate solution and 15 mL of concentrated 
sulphuric acid. Homogenize. Place the tube in a heating block regulated 
at 150°C and adjust the condenser. The attack should be maintained for 
30 min at the same temperature. 

Leave to cool in the air then transfer in a 250 mL wide-necked 
Erlenmeyer flask and bring to approximately 100 mL with washing 
water. Titrate with the 0.2 mol (Fe 2+ ) L" 1 solution (or 0.5 mol L" 1 ) in the 
presence of the indicator and a sequestering agent (cf. "Procedure" under 
Sect. 10.2.2). The dark purple colour will change to luminous green at the 
titration point. 

Controls and Calculation 

- Titration of the Fe 2+ solution (two replicates). 

- Blank titration of prefired quartz by heating under the same conditions 
to correct the thermal destruction of the reagent and to establish the 
effective dichromate concentration (two replicates). 

- Analyse a standard carbohydrate under the same conditions to check 
that oxidation is complete and that a correction coefficient will not be 
needed. 

Without a correction coefficient, total organic C g kg" 1 of 105°C dried 
Kri l=3(10« V) /p 

See "Expression of the Result" under Sect. 10.2.2 for explanation of 
symbols and numbers. 



342 Organic Analysis 

10.2.4 Organic Carbon by Wet Oxidation and 
Spectrocolorimetry 

The French standard NF X3 1-109 (1993) was published for the 
determination of organic carbon by sulfochromic oxidation allowing the 
calculation of the organic matter by means of a multiplying coefficient 
for use in agronomic studies. 

Oxidation is conducted at 135 °C in a thermostated heating block. The 
oxidation of a carbon atom requires the transfer of four electrons. 
Glucose is used as the standard substance and the final determination is 
by absorption spectrometry at 585 nm on aliquots centrifuged for 10 min 
at 2,000 g and filtered to eliminate the suspended particles. 

The method cannot be used in the presence of mineral reducing 
materials (e.g. CI", Fe 2+ ) or of pollution by organic compounds. This 
standard NF X3 1-109 (1993) is referred to in the detailed procedure. 
Notes: 

- After centrifugation, titration of organic C in the medium can be carried 
out at 590 or 625 nm (depending on the author with a standard sucrose). 

- To avoid transfers, a probe spectrometer with optical fibre can be used 
directly in the clarified medium (Baker 1976). 

10.2.5 Total Nitrogen by Wet Method: Introduction 

After carbon, hydrogen, and oxygen, nitrogen is the most abundant 
element in living tissue. It plays a major role in agriculture, nitrogen 
being an essential element for plant growth. In the soil, the organic forms 
can reach approximately 90% of total nitrogen. 

Quantitatively speaking, the total nitrogen value expresses not only the 
N compounds of the organic matter of the soil and biomass (cf. Chap. 
14), but also inorganic nitrogen compounds (cf. Chap. 28). All these 
compounds represent both short- and long-term reserves, i.e. nitrogen that 
is directly or potentially available for plants enabling an improvement in 
yield. 

On the other hand, total nitrogen cannot quantify the values of transfer 
of the different forms of nitrogen between living organisms and inorganic 
materials. Thus total N cannot qualitatively express the diversity of the 
forms of nitrogen that vary considerably in soils subjected to specific 
climatic constraints, environmental conditions such as types of 
vegetation, or different farming systems, nor can it express the complex 
interactions between micro organizations which control the nitrogen 
cycle. The components of proteins, carbohydrates, hemicelluloses, cellulose, 



C, N (H, O, S) 343 

and lignin from living organisms are degraded in the soil, lignin being the 
most stable fraction. 

The phases of N immobilization occur in the form of organic and fixed 
N, and occluded or exchangeable forms of N-NH 4 + . The phases of 
nitrification/denitrification produce N0 2 -, N0 3 -, N 2 , nitrogen oxides 
(with uptake by plant roots and losses through drainage). N processes in 
the soil can be modified by symbiotic associations between plants (e.g. 
leguminous plants) and bacteria (rhizobia, actinomycetes) which involve 
the formation of nodules that enable nitrogen to be fixed from the 
atmosphere. 

The analysis of "Total N" using a wet method derives from that 
proposed by Kjeldahl (1883). Without time-consuming complementary 
treatments that method does not allow all the forms of N pools to be 
recovered entirely. The organic and inorganic nitrogen compounds 
include (in varying proportions): 

-Inorganic forms, (1) NH 4 + -N, which is exchangeable or fixed in the 

mineral or organomineral lattices, (2) N0 3 "N, which is abundant under 

intensive cultivation on heavily fertilized soils, (3) N0 2 ~N, which is 

generally negligible except in waterlogged soils or when it results from 

polluting wastes; N0 3 "-N and N0 2 ~-N are very soluble and are 

consequently easily leached by water infiltration and run-off. 

- Entities whose chemical, physical, and biochemical behaviours are well 

enough defined to enable them to be grouped in selective pools: plant 

fractions in different stages of decay, active microbial biomass, 

biological forms (amino acids, amino sugars, proteins of bacterial 

cells), N subjected to hydrolysis in an acid medium, N of doubtful 

composition not subjected to hydrolysis. Part of N is also included in 

complex humified compounds of varying degrees of stability: relatively 

instable fulvic acids, humic acids with varying degrees of polycondens- 

ation, humins (bound on clays and cementing Fe-Al agents) especially 

protein forms that are weakly attacked by proteases. 

The evolution of soil organic nitrogen is linked to the molecular forms 

of humic compounds. The processes of condensation of humic molecules 

and of formation of organo-mineral compounds modify the stability of 

the different pools. These pools can be ranked on the basis of increasing 

stability as follows: fulvic acids < organo-aluminous compounds < organo- 

ferric compounds < humic acids < various organo-mineral compounds 

< humins fixed on clays. The production of ammonium is an indication of 

instability. 

At the physical level, the organic layers adsorbed superficially on the 
mineral or organomineral matrices react more easily than those fixed in 
the lattices. At the chemical level, the short nitrogen chains are hydrolysed 
more rapidly than the long chains or the N compounds fixed in clays. 



344 Organic Analysis 

The addition of water before analysis releases a varying proportion of 
fixed N by causing swelling of the lattices of certain 2:1 clays. This fixed 
or occluded N can play a role in plant nutrition (Mengel and Scherer 
1981; Keerrthisinghe et al. 1984). Classical Kjeldahl analysis makes it 
possible to quantify only one part of it; so to control this variable, the 
mineral matrix must first be destroyed by a mixture of HF-HC1 (cf. Sect. 

10.3.5 in Chap.10). 

The question of whether it is possible to fully describe the relative 
availability of N compounds in the soil by means of models of the 
chemical and physical compartment that distinguish all the active and 
passive forms has not yet been answered. But whether the answer is yes 
or no, the analysis of total organic and inorganic N is an essential 
component of mathematical models based on a dynamic simulation of the 
forms of N in the soil - plant - climate systems. 

10.2.6 Total Nitrogen by Kjeldahl Method and Titrimetry 
Principle 

Mineralization 

The aim is to transform organic N forms into ammonium-N form using a 
wet method in a concentrated sulphuric acid medium in the presence of 
catalysts. 

(R 3 )N + H 2 S0 4 catalyst ) (NH 4 ) 2 S0 4 + H 2 +C0 2 
2NH 4 + + H 2 S0 4 -^ (NH 4 ) 2 S0 4 + 2H + 

All nitrogen in amide, imide, nitro N-N, nitroso N-O, or other forms is 
transformed into the ammonium salt form. The thermal stability and the 
rise in temperature of the reaction medium are ensured by the addition of 
K 2 S0 4 . Nitrates and nitrites are probably not accounted for, even with a 
mercury catalyst: 

2HN0 3 + 6Hg + 3H 2 S0 4 -* 3Hg 2 S0 4 + 4H 2 + 2NOT 



C, N (H, O, S) 345 



Nitrates and nitrites can be reduced by salicylic acid ^^ and 
sodium hyposulfite Na 2 S 2 3 . 

NH 4 + titration 

The NH 4 + -N produced is transferred to a basic medium by steam 
distillation, and then titrated volumetrically in the presence of an 
indicator. 

2NH 4 OH + H 2 S0 4 -> (NH 4 ) 2 S0 4 + 2H 2 
(NH 4 ) 2 S0 4 + 2NaOH -> Na 2 S0 4 + 2NH 3 T + 2H 2 

After transfer the NH 3 is collected in boric acid (Winkler 1913) and 
titrated by acidimetry: 

4H 3 B0 3 + 2NH 4 OH -> (NH 4 ) 2 B 4 7 + 7H 2 

(NH 4 ) 2 B 4 7 + 3H 2 <^ 2H3BO3 + 2NH 4 B0 2 

2NH 4 B0 2 + 4H 2 <=> 2H 3 B0 3 + 2NH 4 OH 

It is also possible to delay titration, and first to complete distillation, 
and then to perform titration. 

Equipment 

- Analytical balance (±0.1 mg). 

-Rack for attacks with gas heating or thermostatic heating blocks 
regulated at 360°C. 

- 350 mL Pyrex Kjeldahl flasks, or Pyrex cylinders for heating blocks. 

- Dosing spatula for catalysts (= 500 mg, 1 g, 2 g). 

- Glass balls of 6 mm dia. 

- Teflon flask dispenser. 

- Pyrex ball jacks (Fig 10.1a). 

- Fume hood with outlets for heavy vapours. 

- Distillation or steam distillation apparatus. 

- 250 mL Pyrex Erlenmeyer flasks. 

- 50 mL burette (±1/10 mL). 

- Magnetic stirrer with Teflon magnetic stirring bars. 

- 5,000 g centrifuge and 100 mL Pyrex centrifugation tubes. 

- Spectrocolorimeter. 



346 Organic Analysis 

Reagents 

- Sulphuric acid, H 2 S0 4 d= 1.83. 

- Sulphuric acid containing 50 g L" 1 salicylic acid. 

-Catalyst: grind and sieve (Afnor NF22) 100 g of potassium sulphate 
(K 2 S0 4 ), 20 g of copper sulphate (Cu(S0 4 ),5H 2 0), 2 g of grey selenium 
powder (Se); homogenize and store in a wide-necked bottle. 

- 2% boric acid (H 3 B0 3 ) solution in distilled water. 

-Taschiro indicator (Ma Zuazaga 1942): mix one part of 0.1% methyl 
red ethanol solution with three parts of 0.1% bromocresol green ethanol 
solution; store in a brown bottle with dropper. 

-Sodium hydroxide (NaOH) solution =10 molL" 1 : weigh 2.5 kg of 
NaOH pellets and carefully dissolve in 6 L of distilled water; let cool in 
a closed Pyrex bottle (C0 2 is eliminated by precipitation of Na 2 C0 3 ); 
decant Pyrex bottle then discard the bottom part; store in an airtight 
bottle. 

- 1% phenolphtaleine solution in 30% ethanol solution. 

- Standard solutions of 0.1 and 0.05 mol (H+) L 1 sulphuric acid. 
-Standard ammonium sulphate solution: weigh 4.714 g of (NH 4 ) 2 S0 4 

dried on P 2 5 ; dissolve in deionised water and complete to 1 L; lmL 
solution = 1 mg N. 

Procedure (Macro-Method) 

Mineralization 

Weigh 2 g (±0.1 mg) of soil (ground to 125 |Lim particle size) on non- 
gummed cigarette paper (without nitrogen). Close carefully by twisting 
the paper. 

Transfer the sample to a 350 mL glass Pyrex flask. Wet the soil with a 
jet of distilled water from a wash bottle. Agitate gently until complete 
homogenisation of the soils. Leave in contact overnight. 

Carefully (especially with calcareous soils) add 20 mL of concentrated 
sulphuric acid. With a dosing spatula, add 2 g of catalyst. Add 3 glass 
balls (diameter 6 mm), place a ball jack in the neck of the flask and place 
the flask on the rack (Fig. 10.1a). Adjust the gas flame to low and check 
for the formation of foam. The flask should be tilted at an angle of 45- 
60°C to limit the risk of projections and to allow better recovery of 
condensation on the lower walls (Fig. 10.1a). When the soil organic 
matter is broken up, the colour of the sample will have faded; raise the 
heat and boil for 2-3 h without going to dry. 



C, N (H, O, S) 



347 



Distillation and titration by acidimetry 

Leave to cool. Rinse the jack and the flask walls with about 10 mL 
distilled water. Fit the flask in the distillation apparatus (Fig. 10.1b). Add 
100 mL of 10 mol L" 1 sodium hydroxide solution. Turn off the tap to 
begin steam distillation. 

Collect the distillate in the Pyrex Erlenmeyer flask containing 20 mL 
of 2% boric acid solution and 3 drops of Taschiro indicator; take care that 
the end of the exit tube is below the surface of the liquid in the 
Erlenmeyer flask. Approximately 80 mL of the distillate are needed for 
quantitative recovery. 

Titrate by volumetry with the 0.1 or 0.05 mol (H + ) L" 1 sulphuric acid 
solution depending on the estimated quantity of the contents. The end 
point is indicated by a change in colour from green to greyish purple. 



Pyrex jack 

with ball 

M0 mm 




Pyrex 
cylinder 
for foam 
suction 



Reaction medium 
Glass balls 
__ Rack 



> 35 mm 




Fig. 10.1. Minimal equipment needed for Kjeldahl titration of total- N, (a) acid 
mineralization with device to limit acid loss, (b) distillation of resulting 
ammonia by addition of soda; for a more powerful apparatus for steam 
distillation, see Fig. 10.3 in Chap. 14 



Expression of the Results 



The results T N are expressed in mg of nitrogen N per kg of soil dried at 
105°C: 

1 mL H 2 S0 4 0.1 mol(H+)L 1 <=> 0.1 mmol NH 3 is 1.4 mg N. 



348 Organic Analysis 

If V A is the volume (mL) of the 0.1 mol (H + ) L" 1 H 2 S0 4 solution and P 
the mass (g) of the test specimen of soil dried at 105°C, the N content of 

the soil is expressed in mg (N) g" 1 (soil) by: T N = /u • 

Controls 

Controls are made by distilling an ammonium sulphate solution of known 
concentration (1) one distillation after attack under the same conditions as 
the samples in the presence of 1,000°C fired quartz (blank assay), (2) one 
direct distillation of the ammonium sulphate solution. The value of the 
blank assay is calculated by the difference between the two. Two 
reference samples and two replicates of samples chosen randomly in the 
series should be analysed each day. 

Notes 

First the experimental standard X 3 1 1 1 1 (1983) and then the international 
standard NF ISO 11261 (1995) were published for the determination of 
soil total nitrogen by distillation after Kjeldahl mineralization. 

Discolouration is not a sign of the end of mineralization, but an 
indicator of the end of the oxidation of the humified coloured organic 
matter signalling the end of the production of foam. The conversion of 
the N organic products into ammonium obeys other criteria such as 
temperature, catalyst, time, and nature of the N compounds. 

The addition of potassium sulphate enables the boiling point of the 
sulphuric acid to be increased and the attack time to be reduced. To avoid 
losses, use dose less than 0.5 g K 2 S0 4 per millilitre of sulphuric acid with 
selenium as catalyst. The boiling point of H 2 S0 4 , normally 330°C, 
increases to 364°C after addition of 1 g of K 2 S0 4 per mL of sulphuric 
acid. 

Catalysts (1) mercuric oxide is very effective but pollute the 
environment; mercuric oxide can form amino complexes which are not 
released during distillation unless the sample is not treated with 
thiosulfate, (2) selenium allows quantitative recovery of N; the 
temperature should not be too high (<367°C) and attacks should be 
limited in time (maximum 3 h) to avoid the risk of losses, (3) the NF ISO 
11261 standard (1995) recommends replacing Se by anatase Ti0 2 which 
is less polluting for the environment (catalytic mixture 200 g K 2 S0 4 , 6 g 
CuS0 4 ,5H 2 0, 6 g Ti0 2 ). 



C, N (H, O, S) 349 

Grinding the samples to 125 |Lim enables micro-methods to be used 
without a serious reduction in precision (Parnas and Wagner, 1921; 
Markham 1942), thereby reducing the cost price, pollution, and the extent 
of work surface required. 

The use of heating blocks decreases the turbulence of the attacks as 
heating is more regularly distributed. Programming the stages of heating 
makes it possible to accomplish the mineralization cycle without 
monitoring. 

10.2.7 Kjeldahl N, Titration by Spectrocolorimetry 



Principle 

After digestion in a sulphuric acid medium catalysed by selenium 
(without copper or titanium) the reaction (Berthelot 1859; Bolleter et al. 
1961) is continued in basic medium (sodium hydroxide buffer and dibasic 
sodium phosphate). 

The NH 4 + ion reacts with the sodium hypochlorite and the sodium 
phenolate (or the sodium salicylate) to give a blue-green complex. The 
reaction is catalysed by the sodium nitroprussiate. 

// \ // \ Indophenoljaune 



NH 3 + CIO"— NH 2 C1 ^ ^"^n-ci — -o=( J=n^^oh 

Bleu d'indophenol 

OH" 



Equipment 

- cf. "Equipment" under Sect. 10.2.6 

- Centrifuge 

- Automated segmented continuous-flow analysis with NH 4 + manifold 

Reagents 

All the reagents should be of reference analytical grade: 

- Mineralization products: see "Reagents" under Sect. 10.2.6. 

- Brij 35 detergent: polyoxyethylene lauryl ether C^ItsCOCIfcCiyOH 
(to limit contamination of the analytical manifold). 

- 20% sodium hydroxide (NaOH) stock solution. 

- Sodium phosphate, Na 2 HP0 4 ,2H 2 0. 



350 



Organic Analysis 



20% sodium potassium tartrate, NaKC4H 4 06,4H 2 (Seignette salt) 

stock solution. 

Stock buffer solution: dissolve 89 g Na 2 HP0 4 ,2H 2 in 800 mL 

deionised water; cool and add 50 mL of 20% NaOH solution; 

homogenize and complete to 1,000 mL. 

Buffer solution: mix 200 mL of buffer stock solution, 250 mL of stock 

solution and 20% of sodium potassium tartrate; add 60 mL of 20% 

NaOH stock solution and about 300 mL water; homogenize and cool; 

add 1 mL of Brij 35; bring to 1 L and homogenize. 

Sodium salicylate: dissolve 150 g of sodium salicylate and 300 mg of 

sodium nitroprussiate in 800 mL water; bring to 1 L; filter on a 

Buchner funnel with a blue filter without NH 4 and add 1 mL of Brij 35; 

store in a brown bottle protected from the light. 

Sodium nitroprussiate, Na 2 Fe(CN) 5 NO,2H 2 0. 

Sodium hypochlorite, NaCIO: add 5 mL NaCIO in 80 mL distilled 

water, bring to 100 mL; add 2 drops of Brij 35; prepare a fresh solution 

each day. 

Ammonium sulphate, (NH 4 ) 2 S0 4 . 

Hydrochloric acid, HC1 37%. 

Standard NH 4 : pour 80 mL HC1 37% in 800 mL water; cool and bring 

to 1,000 mL with water; prepare a stock solution of ammonium 

sulphate at 100 mg NH 4 + -N mL" 1 ; store in the refrigerator; solutions 

should be freshly prepared each day starting from the stock solution to 

provide ranges from to 50 jLXg mL" 1 . 



Procedure 



Flows 
mLmn 1 



Inputs 



Washing 



2.00 Water 



— m A T A/V v T A/VV^ 



Water bath 
37°C 
Visible 
spectrometer 

— ► 



0.32 
1 1.00 



Air 



Buffer solution 



0.10 Sampl e 



0.32 Salicyl.+nitropr. 



0.16 NaCIO 



1.20 Cell 



output 



Waste 



1 5 mm cell 
660 nm filter 



Fig. 10.2. Titration of total nitrogen by segmented continuous-flow analysis after 
mineralization (Buurmans et al. 1996) 



C,N(H,0, S) 351 

Mineralization is carried out as in "Mineralization" in "Procedure 
(Macro-Method)" under Sect. 10.2.6, but with no copper sulphate in the 
catalyst mixture. At the end of the attack, cool the sample, and after 
complete cooling, dilute and pour into a 250 mL volumetric flask. 

After adjusting the volume and homogenisation, centrifuge each 
aliquot at 3,000 g for 15 min to obtain a clear solution. Remove the test 
samples for analysis from the supernatant and dilute if necessary in 
suitable stages of dilution (for example five times) when introducing the 
sample in the N-NH 4 manifold. Titration is by absorption at 660 nm in a 
15 mm colorimetric cell. The manifold proposed by Buurmans et al. 
(1996) is shown in Fig. 10.2 without the dilution stage. In the case of 
highly calcareous soils, it is sometimes necessary to add EDTA in 
addition to K and Na tartrate to avoid side effects due to the calcium 
which is precipitated in the sulphuric acid medium and can give a 
colloidal precipitate that is not easily visible to the naked eye 
(Gautheyrou and Gautheyrou 1965). 

10.2.8 Kjeldahl N, Titration by a Selective Electrode 



Principle 

The principle is similar to that described in Sect. 10.3.2 of Chap. 28 for 
the titration of ammonium nitrogen. Only the conditions of use are a little 
different. Here the gas diffusion electrode enables the results to be read 
directly on the calibration curve. The response is linear in the 0.5-500 mg 
L" 1 zone of concentration of nitrogen with a lower degree of precision for 
high concentrations because of the logarithmic response. A range of 
concentrations of from 0.5 to 10 mg L" 1 is recommended. 

This method has the advantage of being extremely simple and a wide 
range of concentrations can be analysed. Daily calibration is not 
necessary if the working temperature is always the same. Under normal 
working conditions, no interference occurs. 

Equipment 

- Mineralization equipment: cf. "Equipment" under Sect. 10.2.6. 

- Orion model 95-100 gas electrode. 

- Ionometer (pHmeter, mVmeter) with a resolution of 0.1 mV. 

- Thermostated bath regulated at 25°C. 

- Magnetic stirrer. 



352 Organic Analysis 

Reagents 

- Mineralization acid: cf. "Reagents" under Sect. 10.2.6. 

- Ten mole (NaOH) L" 1 soda solution. 

- Standard solution: dissolve 1.179 g of ammonium sulphate ((NH 4 ) 2 S0 4 , 
mw= 132.12) in distilled water and bring to 250 mL; this solution 
contains 1 mg (N) mL" 1 ; dilute 10 times to obtain the initial reference 
solution 0.1 mg (N) mL" 1 . 

- Ammonium chloride solution to fill and maintain the electrode: 0.1 mol 
(NH 4 C1) L 1 . 

Procedure 

Mineralization is carried out as in "Mineralization" in "Procedure 
(Macro-Method)" under Sect. 10.2.6. At the end of the attack, cool the 
sample and transfer it to a 200 mL volumetric flask. After complete 
cooling, bring the volume to 200 mL. 

Construction of the standard curve: in 50 mL beakers add the volumes 
of the 0.1 mg (N) mL" 1 reference solution listed in Table 10.4; complete 
to 20 mL with additional volumes of the blank attack solution (cf. Sect. 
10.2.). 

Bring the temperature of the beakers and the blank to 25°C, immerse 
the electrode in the blank avoiding the presence of air under the 
membrane; add 2 mL of the 10 mol (NaOH) L" 1 soda solution; start 
gentle agitation to limit the Vortex effect; after 5 min, adjust the zero of 
the ionometer. 

Table 10.4. Range of titration of total nitrogen by ionometry; the content of the soil 
is given for a test sample of 2 g, volume of solution after attack (cf. 
"Mineralization" in "Procedure (Macro-Method)" under Sect. 10.2.6): 
200 mL, aliquot for titration: 20 mL 



mL of standard 


mL of attack 


mL NaOH 


solution 


soil 


solution 0.1 


solution for 


10 mol L -1 


concentration 


concentration 


mg (N) mL" 1 


20 mL 




mg (N) L" 1 


mg (N) g" 1 


1 


19 


2 


5 


0.5 


2 


18 


2 


10 


1 


4 


16 


2 


20 


2 


6 


14 


2 


30 


3 


8 


12 


2 


40 


4 


10 


10 


2 


50 


5 



Proceed in the same way for the different points of the calibration 
range. Record the signal in mV and plot the calibration curve with the 
logarithms of nitrogen concentrations on the x-coordinate and mV on the 
^-coordinate. 



C, N (H, O, S) 353 

The measurements should be carried out on 20 mL aliquots of each 
attack solution using the same technique as for the calibrations. It is 
essential to carry out titrations immediately after the addition of soda to 
avoid loss of ammonia. 

Remarks 

Before starting analysis, it is important to check the pH of the 
measurement solutions which must be from 1 1 to 13 after the addition of 
soda (Table 10.4). In the case of a lower pH, slightly increase the volume 
of the 10 mol (NaOH) L" 1 soda solution. 

After use, the electrode should be stored carefully following the 
manufacturer's instructions. The filling solution should be renewed 
periodically. 

10.2.9 Mechanization and Automation of the Kjeldahl Method 

The Kjeldahl method has the advantage of requiring only simple 
equipment and of being the reference method. However, manual methods 
are time consuming. They require large laboratory work surfaces and 
fume hoods to evacuate the heavy vapours. The technique can be 
improved by automation which makes it possible to avoid direct handling 
of dangerous reagents such as boiling sulphuric acid or concentrated 
soda. 

The use of programmable heating blocks (e.g. Technicon, Skalar, 
Tecator) enables the temperatures of mineralization to be regulated and 
sudden starts and foaming during the attacks to be limited. 

Partially automated equipment exists comprising management stations 
for mineralization, distillation, and titration at the macro and micro scale 
(e.g. Bicasa, Bucchi, Gerhart, Skalar, Foss-Tecator, Velp). Prolabo, Cem, 
Questron enable accelerated mineralization by microwave heating; the 
attack containers are processed automatically. 

Complete automation was provided by manufacturers such as Tecator, 
Foss, Perstop, or Gerhart. Mineralization is automated, and final titration 
is carried out by titrimetry with potentiometric detection of the end point 
of titration, or by spectrocolorimetry. The job of the analyst is limited to 
filling up the reagents, regulating the heating programmes and recovering 
the results at the end of the day. Monitoring is simplified because an 
alarm goes off in the case of accident. 



354 Organic Analysis 

10.2.10 Modified Procedures for N0 3 ", N0 2 ", and Fixed N 

These methods are seldom used in repetitive analyses because of the 
uncertainty of the results, the length of time required for the procedures, 
and the fact that the contents are often not very significant. 

Nitrate and Nitrite 

The determination of total N using wet methods does not take nitrate into 
account, but as it is generally not present in large quantities in natural 
environments, there will be no effect on results obtained on dried 
samples. However, in the case of cultivated soils dressed with manure 
with a high N content, it is preferable to rapidly check for the presence of 
nitrate using commercial tests, because nitrate and nitrite can introduce a 
random error. 

Nitrate and nitrite can be included in Total-N by means of redox 
sequences before Kjeldahl mineralization (1) by KMn0 4 /Fe 2+ where the 
nitrite is oxidized into nitrate by a potassium permanganate solution, 
the nitrate then being reduced in ammonium by ferrous iron before the 
mineralization sequence in the traditional method (cf. "Procedure 
(Macro-Method)" under Sect. 10.2.6); (2) by a system using salicylic 
acid/ammonium thiosulfate in which nitration by salicylic acid can occur 
quantitatively only in absence of water (Fuson 1962); the nitrated 
compounds are then reduced by ammonium thiosulfate before Kjeldahl 
mineralization as in "Procedure (Macro-Method)" under Sect. 10.2.6 
(Nelson and Sommers 1980; Du Preez and Bate 1989). The international 
NF ISO 11261 standard (1995) recommends (1) action of 4 mL of a 
salicylic/sulphuric acid mixture (25 g salicylic acid in 1 L concentrated 
H 2 S0 4 ) for a few hours or overnight, (2) the addition of 0.5 g of sodium 
thiosulfate with moderate heating until the foam disappears, (3) cooling 
the flask and the addition of a catalyst (dioxide of titanium instead of 
selenium) and sulphuric acid for Kjeldahl mineralization. Nitrate and 
nitrite can also be titrated separately using the methods described in 
Chap. 28. 

Fixed or Occluded N 

The determination of fixed or occluded N is very delicate. Indeed, some 
authors have raised questions both with respect to its nature (inorganic 
NH 4 + -N only, or organic and inorganic N) and to its modes of fixing N. 
Fixed N is not titrated quantitatively by the traditional method particularly 
in soils containing 2:1 clays. To titrate fixed N, a polyethylene bottle is 
used, and the clay lattice is destroyed by a mixture HF-KC1 or concentrated 
KOH (cf. Sect. 28.3.5 of Chap. 28). The organic digestion of N is then 



C, N (H, O, S) 355 

carried out in traditional Kjeldahl flasks (cf. Sect. 10.2.6). 

Remarks 

As the glass of the Kjeldahl flasks is seriously affected, their quality 
deteriorates rapidly and they need to be regularly replaced. The fixing of 
N-NH 4 in the mineral lattices is more important in the deep horizons 
which are rich in clay and low in N compounds; the increase in N-fixing 
power as a function of the nature of clays can be expressed by: 
vermiculites > illites > montmorillonites > kaolinites. 

In certain cases the expansion of the lattices, which is obtained by the 
addition of water, can lead to higher values for total -N (Bal 1925). 
Conversely, the rate of recovery of NO3-N may be lower (Bremner and 
Mulvaney 1982). 



10.3 Dry Methods 



10.3.1 Total Carbon by Simple Volatilization 

These methods are based on oxidation of the soil organic matter and on 
destruction of carbonates by heating of the samples to relatively high 
temperatures for a given length of time. Measurement of the loss in mass 
(cf. Chap. 1) allows estimation of gaseous losses that are mainly in the 
form of C0 2 and H 2 0. Resistance or induction furnaces can function at 
temperatures of 1,000-1, 500°C. 

In the simplest case, an open air circuit is used, possibly with oxygen 
enrichment (low temperature ashing, LTA), or in closed systems with 
controlled temperature and time. Traps can be used to block some 
compounds, and catalysts can accelerate the reactions. Gas separation 
systems of varying degrees of sophistication are used in sequential 
analyses of evolved gas. To sum up, a temperature range lower than 
500°C enables collection of the C0 2 released by the organic matter and 
temperatures higher than 500°C, of C0 2 produced from carbonates. 

Although calcination methods using an open circuit for the 
determination of Total-C are very simple to implement, they cannot be 
regarded as quantitative and generally have be supplemented by thermal 
analysis techniques (cf. Chap. 7). In an oxidizing atmosphere, gravimetric 
measurements will represent: 

- Organic C transformed into C0 2 . 

- Inorganic C starting from approximately 400°C. 



356 Organic Analysis 

-Different forms of water (hygroscopic, interstitial, water of 

crystallization, hydroxyl groups) which are moved throughout the 

thermal cycle. 
- Volatile forms of N, S, certain metals, and metalloids (CI). 

These displacements vary with the temperature used and with the times 
of application. Still other transformations can disturb measurements. 

In an oxidizing atmosphere, some compounds of the soil that are 
sensitive to redox reactions will increase in weight while passing to a 
higher valence, for example Fe 2+ — > Fe 3+ . Sulphur compounds can, by 
recombination, show simultaneous losses and increases in mass as a 
function of the medium: H 2 S -» S0 2 -» S0 3 -» S0 4 2 ". 

The results are thus random, and are related to the atmosphere used 
(oxidizing, neutral, reducing) and to effects of the matrix. The 
thermostable residue of the soil can be considered reached between 1,100 
and 1,600°C. In most cases, total organic and inorganic C with C 
obtained by the simple measurement of the losses on ignition cannot be 
directly assimilated. Below 500°C, C losses at lower temperatures can 
easily characterize oxidizing C, but it is difficult to quantify this variable. 

However, certain soils enable results with a reduced margin of error, in 
particular sandy soils with low 1:1 clay content, but not calcareous soils, 
or soils rich in organic matter. The decarboxylation of the organic matter 
starts around 180°C, and at 250 °C, it is estimated that 70% of organic C 
is oxidized, and 90% around 500°C. Temperatures between 800 and 
950°C are needed to obtain 99% of oxidation of organic C. 

In the case of carbonates, decomposition starts at around 400°C: 
calcium and magnesium carbonates (e.g. calcite-aragonite, magnesite, 
dolomites) generally display the same type of thermal behaviour, but 
biogenic calcite (shells, skeletons, calcareous debris), iron carbonate 
(siderite), fossil coals, and resins can respond differently, as can sodium 
carbonates with a low melting point (851°C). 

10.3.2 Simultaneous Instrumental Analysis by Dry Combustion: 
CHN(OS) 



Principle 

Thanks to improved equipment, the purchase of entirely automated 
CHN(OS) apparatuses (Pansu et al. 2001) is very tempting. These 
apparatuses are very profitable in laboratories that do many repetitive 



C, N (H, O, S) 357 

analyses, as they can do simultaneous analysis of C, N, and H and, under 
adequate operating conditions, of O and S in addition. To ensure the 
uninterrupted performance of the apparatuses, the operators need to be 
specially trained. The apparatuses also have to be regularly checked and 
maintained, and the exact conditions of use clearly defined. In this type of 
set-up, each element in the chain is linked to the others (furnace, CuO, 
catalysts, and different traps) and the weakest link in the chain determines 
the final quality of the analyses. The method is the subject of the 
international standard NF ISO 10694 (1995) for the analysis of organic 
carbon and total carbon of soils. 

Seal a given mass representative of the sample in a tin (or silver) 
micro-capsule and place it in a furnace at 1,100°C in a controlled 
atmosphere in the presence of oxygen and copper oxide. The different 
forms of organic and inorganic C and N oxidize or break down rapidly 
according to the principle of the Liebig reaction (1830) 

C + 2CuO ° 2 ' 1 ' 8QQ ° C ) C0 2 T + 2Cu 

The temperature of 1,800°C is reached by flash combustion of the tin 
capsules. The carbonates and bicarbonates are broken up simultaneously. 
For titration of organic C, carbonates, and bicarbonates must be 
eliminated chemically before the sample is placed in the furnace 

CaC0 3 1 ' 0QQ ° C ) C0 2 T + CaO (thermal destruction) 

CaC0 3 + 2H + -> C0 2 T + H 2 + Ca 2+ (chemical destruction) 

If methane is present and has to be titrated, Cr 2 3 should be used to 
ensure perfect oxidation. The organic nitrogen that is oxidized during 
combustion gives nitrogen oxides which must be reduced with copper to 
transform all nitrogen oxides into the N 2 form by the Dumas (1831) 
method. Inorganic N compounds (NH 4 + , N0 3 -, N0 2 -) are subtracted to 
obtain total organic N. Different catalysts can be used to accelerate the 
reactions or to make them quantitative. 

Finally, the gas products are identified in suitable detectors; the 
gaseous compounds can be separated on temporary specific traps or by 
gas chromatography, depending on the system used. In soils that do not 
contain carbonates or bicarbonates, or have not received calcareous 
amendments or lime: 

- Inorganic C = 0. 

- Total C = total organic C. 



358 Organic Analysis 

Equipment 

- Elementary Analyzer CHN(OS), preferably able to handle samples of 
approximately 100 mg or more to compensate for the heterogeneity of 
carbon in soil (Pansu et al. 200 1) 1 . 

- Micro analytical balance. 

- Needle-nosed pliers to close the capsules. 

- Micro pipette adjustable 0.1-0.5 mL. 

- Ceramic plate for treatment of calcareous samples. 

- Controlled temperature hotplate. 

Products 

All consumables should be suitable for the procedure and type of 
analytical materials used (analytical grade reference products): 

- Combustive gases, carrier gas purity: 99.98% 

- Copper oxide, (CuO) 

- Copper in the form of wire or chips (Cu) 

- Magnesium perchlorate (Mg(C10 4 ) 2 , ascarite (NaOH-asbestos), Mn0 2 , 
etc. for traps 

- Tin or silver micro capsules, capacity 100 mg 

- A range of catalysts 

- Standard substances for calibration and control such as acetanilide 
(C 8 H 9 ON, 71.09% C, 6.71% H, 10.36% N, 11.84% O), atropine 
(C17H25NO3), sulphanilamide H 2 NC 6 H 4 S02NH2, picric acid (2,4,6 
trinitrophenol (N0 2 )3C 6 H 2 OH), thiourea (H 2 NCSNH 2 ), etc. 

Procedure 



Non-calcareous soils 

All weighing should be done on analytical balances (± 0.1 or ± 0.01 mg) 

Calibration: weigh 5 sample specimens of increasing mass of a 
standard in the range of concentration accepted by the CHN apparatus 
concerned. The precision of the equipment should be tested by comparing 
the theoretical contents with the contents obtained. 



A higher mass of sample specimen increases the representativeness of the 
sample but produces more ashes in the combustion furnace. The current trend 
is to reduce the mass of sample specimen to about 10 mg and to increase the 
homogeneity of the sample by very fine grinding to a particle-size of less than 
100 micrometers. 



C, N (H, O, S) 359 

Samples: grind the samples to 125 |Lim (Sieve AFNOR NF22) and dry 
for 48 h in a P 2 5 desiccator to limit saturation of the traps. In tin or 
silver capsules, carefully weigh from 50 to 100 mg of sample (depending 
on the estimated contents of C and N) dried on P20 5 , and seal the capsule. 
Place the capsules in the sample distributor of the CHN apparatus. 

At the same time, weigh 1 g of the same sample to measure residual 
moisture by drying at 105°C in order to be able to correct the results (cf. 
Chap. 1). Direct drying of the samples at 105°C before CHN analysis 
may increase ammonium fixation in the lattice of 2:1 clays, but in most 
case this is acceptable. 

Note 

Andosols and histisols pose two problems (1) the residual moisture of air- 
dried soil is very high, up to 50-60% after 6 months; (2) low bulk density 
can reach 0.30-0.25. In this case it is not possible to weigh 100 mg in the 
capsules. 

Carbon in calcareous soils 

In the case of calcareous samples or soils that have been limed, if there is 
no siderite (not easily decomposable FeC0 3 ), treatment with 10% 
hydrochloric acid solution is sufficient to destroy the carbonates and 
bicarbonates in the sample: 

- Weigh 100 mg of sample in a silver capsule. 

- Place the open capsule on a ceramic plate. 

- With a micro pipette, slowly add 0.1-0.2 mL of 10% HC1 depending on 
the estimated quantities of carbonate and bicarbonate. 

-Leave in contact for 2 h then add 0.1 mL of the 10% HC1 solution. 
Check that gaseous emission is complete. 

- Place the ceramic plate on a hotplate at 60°C and bring to dry. Leave to 
cool and seal the capsule. Place the capsule on the CHN sampler to 
measure total organic C. A second sample can be weighed without 
destroying carbonates to measure total C. Total inorganic C can be 
calculated by difference. 

On certain apparatuses it is preferable to use phosphoric acid rather 
than hydrochloric acid to limit the effects of chloride on the catalysts and 
on the traps. 



360 Organic Analysis 

Calculations 

The results are calculated directly and printed by the CHN analyser after 
measurement of the surface of the C and N peaks. These calculations take 
the weight of the sample into account. 

To determine total organic nitrogen, the results obtained for total 
inorganic nitrogen have to be taken into account (cf. Chap. 28). 
Total N (CHN) = inorganic Nitrogen + organic Nitrogen 

Titration of Hydrogen, Oxygen and Sulphur 

Hydrogen is titrated together with carbon and nitrogen using the 
procedure described in "Procedure" under Sect. 10.3.2 by measuring the 
water formed during combustion. However this measurement is difficult 
to interpret in most soils because hydrogen resulting from combustion of 
the organic molecules and hydrogen coming from the different forms of 
water (e.g. adsorption, hydration, and hydroxylation) is not separated in 
the H 2 signal. In soils that are not very clayey and when using carefully 
dried samples, the H 2 signal can represent only H coming from 
combustion of the organic molecules. The C:H ratios then reveal the 
stages of evolution of the soil organic matter. 

The titration of oxygen and sulphur is generally carried out with 
standard CHN equipment by simply modifying the instrumental 
parameters. For sulphur, controlled oxidation is used, generally in the 
presence of tungstic anhydride. For oxygen, pyrolysis is used instead of 
combustion (the oxygen supply is cut off) on a sample specimen reserved 
for this measurement. 

Total organic oxygen: the sample is subjected to pyrolysis at 1,100°C 
in the presence of a catalyst such as a mixture of carbon and nickel. 
Carbon monoxide (CO) is formed, then isolated by a separation system 
(generally with a trap or by chromatography) and quantified by a system 
of detection (e.g. a catharometer). Oxygen is determined by comparison 
with standards of known O content in the same way as for C and N 
described in "Procedure" under Sect. 10.3.2: one molecule of CO 
represents one O atom in the sample. 

Total organic oxygen corresponds to only a small fraction of total 
oxygen of the soil. Inorganic oxygen, which is more abundant, is 
generally estimated starting from the total analysis of the major elements 
(cf. Chap. 31), by calculating the difference between the sum of the 
contents expressed in oxides and the sum of the contents expressed in 
elements. 

Total sulphur, the sample is oxidized at 1,100°C in the presence of 
tungstic anhydride W0 3 or a mixture of tungstic and vanadic oxides. 



C, N(H,0, S) 361 

Oxides of sulphur (e.g. sulphur trioxide S0 3 , sulphur dioxide S0 2 ) are 
formed. A stage of reduction on a copper column transforms all oxides 
into the S0 2 form. This gas is then isolated and titrated using a system 
that will depend on the type of equipment used (traps, chromatography) 
and the type of detector (catharometer, IR detector): one molecule of S0 2 
corresponds to one atom of S in the sample. Depending on the type of 
equipment used, titration of sulphur may or may not be carried out 
simultaneously with that of carbon and nitrogen. 

Instrumental CHNS methods give a value for the sulphur content that 
can generally be regarded as the total sulphur content of the soil: 
oxidation of organic sulphur and sulphides, decomposition of most of the 
sulphates (Laurent 1990). However, care should be taken given the 
diversity of natural forms of sulphur. For a more detailed analysis of this 
element, see Chap. 30. 

Remarks 

Alkaline salts (e.g. sodium carbonate, chlorides, phosphates) melt to a 
varying degree and delay the oxidation of C into C0 2 by coating the 
organic particles and disturbing the cycle of analysis. 

Salts that are volatile at 700°C or more (e.g. some chlorides, bromides, 
or iodides) can contaminate (or corrode) the circuits, traps, and catalysts. 

Magnesium perchlorate Mg(C10) 4 ) 2 , sold under the name of anhy drone 
or dehydrite, is used to trap water. It is a relatively unstable product that, 
after use, must be eliminated in the same way as other dangerous waste 
by the laboratory. 

Residues of calcination in the furnace have to be removed frequently, 
particularly in the case of carbonated saline soils. Changing the protective 
nacelles makes it possible (1) to preserve the quality of the filters for 
elimination of the fine particles likely to be present in the gas phase and 
(2) to limit the retention of volatile products of combustion. Traps, 
compounds, catalysts, and filters must be changed regularly, respecting 
the change-by deadlines. Most breakdowns and doubtful results are due 
to inadequate maintenance. 

The use of synthetic standards can result in errors, as their physical and 
chemical behaviour may differ from that of soil organic matter. However, 
the temperature of 1,800°C reached during the flash combustion of the 
capsules makes it possible to release all the organic compounds. Soil 
standards with certified organic-C contents are also available for 
calibration. Elementary CHN(OS) analysers generally give accurate 
results but these may be higher than wet oxidation. 



362 



Organic Analysis 



10.3.3 CHNOS by Thermal Analysis 

In detailed studies it is important not to overlook the possibilities offered 
by instrumental methods such as differential thermal analysis (DTA) and 
thermogravimetric analysis (TGA), coupled or not with measurements of 
evolved gas analysis (EGA) (cf. Chap. 7). 



Oxidation of 
nucleus of 
humic acids 



Water losses 
from gypsum 
allophane 



Water losses 
from clays, 
oxidation of 
functional groups 
of humic acids 



j-L 



Biogenic 
calcite 



Calcite 



Magnesite — Aragonit!e 



J_l L. 



100 300 

Mostly oxidation 
of organic C 



500 



700 



900 T°C 



Mostly decomposition of 
inorganic C 



Fig. 10.3. Oxidation of organic C and decomposition of inorganic C as a function 
of temperature 



Controlled pyrolysis (with suitable temperature/time programmes) 
enables oxidation of the organic matter and the decomposition of the 
inorganic compounds to be monitored. It is possible to identify and 
quantify the nature of the gases (e.g. C0 2 , CO, CH 4 , H 2 0, NH 3 , H 2 S, 
S0 2 ) that correspond to the DTA and TGA peaks as a function of 
temperature, to monitor the decomposition or the transformation of 
products containing C and N, and to separate exothermic and 
endothermic peaks of H 2 and C0 2 , etc. 

For more complete studies, trapping of certain phases at -180°C, then 
their separation by gas chromatography, or by coupling with, for 



C, N (H, O, S) 363 

example, infra-red or mass spectrometers detectors, enables detailed 
characterization of soil organic matter. 

The sum of the different carbon phases gives total C; and it is possible 
to roughly separate total organic C and total inorganic C (Fig. 10.3). In 
studies of organic geochemistry, the molecular mass of the fragments of 
pyrolysis is measured in a mass spectrometer with discontinuous 
injections. The structure of complex substances with high molecular 
weights can be characterized. 

10.3.4 C and N Non-Destructive Instrumental Analysis 

Analysis by diffuse reflectance near infra-red spectrometry (MRS, cf. 
Chap. 5) is carried out directly on the soil sieved on an AFNOR NF22 
sieve (125 |Lim mesh). 

Take a sample weighing approximately 1 g after drying in the drying 
oven and carefully pack it in a special cup and place the cup in the 
measuring chamber. The surface of the sample must be perfectly flat. 
Each component of the organic complexes of the soil has a specific 
absorption point between 700 and 2,500 nm in the MR spectrum, due to 
the vibrations of stretching and deformation of the inter-elements bonds 
(cf. Sect. 10.3.1 in Chap. 5). 

For C and N measurement, information provided by the NIRS spectra 
must be calibrated to data from other methods of measurement. Then the 
calibration curve is used to quantify C and N in unknown samples. Good 
calibration has been obtained with the methods described in Sects. 
10.3.1-10.3.3 above (Krishnan et al. 1980; Dalai and Henry 1986; Morra 
et al. 1991; Fidencio et al. 2002). The method is non-destructive and the 
samples can be used for other measurements. As each apparatus has its 
own particular characteristics with regard to selection and optimization, 
the manufacturer's recommendations should be followed (e.g. Bran- 
Luebbe, Bruker, Foss, Leco, Nicolet, Perkin-Elmer, and Perstorp). 

Rapid methods to estimate soil C in the field are currently the object of 
serious investigation in research programmes dedicated to C 
sequestration in soils as part of the effort to decrease emissions of 
greenhouse gases in the atmosphere. Though they are less precise than 
laboratory techniques using a wet method or dry combustion, these 
methods have the advantage of rapidly providing a very large number of 
measurements at a lower cost. MRS methods for the processing of 
analytical signals have been developed thanks to spectacular progress in 
software. This software can provides quantitative data based on spectra 
that chemists previously found very difficult to interpret. 



364 



Organic Analysis 



In addition to the MRS method, the laser induced breakdown 
spectroscopy method (LIBS) has been proposed for soil carbon (Cremers 
et al. 1996). The detection limit is approximately 300 mg kg" 1 , precision 
4-5%, accuracy 3-14%, and the speed of analysis is more than one 
sample per minute (Cremers et al. 2001). 

1 0.3.5 Simultaneous Analysis of the Different C and N Isotopes 



i 



i i 



5 



* 



=5^ 



Wet 
oxidation 




93.2% 



Dry combustion 



■ -e C_ 



£ 



Pump for flow 
partitioning 



i 



Traps for 
measurement 

of C0 9 - 4 C 



5.2% 



1.6% 



Analysis 
oftotal-C 



Fig. 10.4. Simultaneous determination of total- and C-carbon (diagram by P. 
Bottner, CEFE-CNRS Montpellier, France, personal communication), 
(a) soda lime for 2 purification, (b) boiling liquid sample + K 2 Cr 2 7 , (c) 
combustion tube, (d) post combustion tube (CuO), (e) solid sample, (f) 
900°C combustion furnace, (g) automated burette containing H 2 S0 4 + 
H3PO4, (h) purification traps (water condensation, chromic and acid 
foams), (i) columns with glass balls impregnated with ethylene glycol 
mono-ethyl ether + mono-ethanolamine to trap C0 2 , (j) soda lime traps 
for security, (k) ventilated external output, (I) output of excess 2 

Dry combustion analysers - CHN(OS) - can be coupled with mass 
spectrometers enabling the study of the different isotopes of the elements, 
particularly 14 C, 13 C, 15 N (Pansu et al. 2001). Often 15 N nitrogen is also 
measured starting from the Kjeldahl distillates in the form of ammonium. 
The carbon dioxide of the effluent combustion gas can also be trapped for 
later determination of the isotopes. The Bottner and Warembourg 



C, N (H, O, S) 365 

equipment (1976), shown in schematic form in Fig. 10.4, was used for 
more than 20 years for simultaneous titration of total-C (carmhograph 
12 A) and 14 C-carbon (liquid scintillation) starting from solid (soils, 
plants) or liquid (water of the soil) samples and proved to be reliable. 



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Stewart RA and Porter LK (1963) Inability of Kjeldahl methods to fully measure 

indogeneous fixed ammonium in some soil. Soil Sci. Soc. Am. Proc, 

27,41-43. 



370 Organic Analysis 

Szekely E (1991) A rapid colorimetric method for analysis of nitrate nitrogen by 

reduction to nitrite. Commun. Soil Set Plant Anal., 22, 1295-1302. 
Tabatabai MA and Bremner JM (1991) Automated instruments for determination 

of total carbon, nitrogen and sulphur in soils by combustion techniques. 

In Soil analysis - Modern instrumental techniques, Smith KA ed. 

Marcel Dekker, NY, USA 261-286. 
Takesako H (1991) Double-plunger pump system flow injection 

spectrophotometric determination of inorganic nitrogen in soil extracts, 

part 1, flow injection analysis of ammonium nitrogen in soil extracts, 

part 2, flow injection analysis of nitrate nitrogen in soil extracts, Jpn J 

SoilSci. Plant Nut., 62, 128-134, 135-140 
Tel DA and Jansen J (1992) Determination of total nitrogen in soil digest using a 

Traacs 800 Autoanalyser. Commun. Soil Sci. Plant Anal., 23, 2729- 

2736. 
Tisley J (1950) Determination of organic carbon in soils by dichromate mixtures 

in Trans. 4 th Int. Cong. Soil. Sci., 1, 161-169. Hoitsemo Brothers, 

Groningen. 
Verardo DJ, Froelich PN and Mcintyre A (1990) Determination of organic 

carbon and nitrogen in marine sediments using the Carlo Erba Na. 1500 

analyser. Deep Sea Res., 37, 157-165. 
Volonteri HJ (1983) Modificacion de un metodo colorimetrico para la 

determinacion del nitrogeno total en suelos y pastos. Ciencia del suelo, 

1,98-99. 
Walinga J, Kithome M, Novozamsky I, Houba VJG and Vanderlee J J (1992) 

Spectrometric determination of organic carbon in soils. Commun. Soil 

Sci. Plant Anal, 23, 1935-1944. 
Winter JP, Gregorich EG, Voroney RP and Kachanoski RG (1990) Comparison 

of two samples oxidation methods for quantitative measurement of 12 C 

and 14 C in plant and soil. Canad. J. Soil Sci., 70, 525-529. 
Yeumans JC and Bremner JM (1991) Carbon and nitrogen analysis of soils by 

automated combustion techniques. Commun. Soil Sci. Plant Anal, 22, 

843-850. 



11 



Quantification of Humic Compounds 



11.1 Humus in Soils 



11.1.1 Definition 

Humus plays a fundamental role in ecological processes as a source of 
carbon for the atmosphere, a sink of carbon for the biosphere, a sink and 
source of fertilizers for plants, and a factor that influences soil properties, 
and important reviews are regularly devoted to it (Kononova 1966; Flaig 
et al. 1975; Schnitzer 1978; Stevenson 1982; 1994; Aiken et al. 1985; 
Tate 1992; Carter and Stewart 1995; Piccolo 1996; Magdoff et al. 1996; 
Hessen and Tranvik 1998). 

Stevenson (1982) defined the term "humus" (or humified matter) as the 
sum of organic compounds in the soil with the exclusion of living 
organisms in the biomass and non-decomposed or partially decomposed 
organic debris of plant or animal origin (cf. Chap. 9). The use of the term 
"soil organic matter" is less clear: the term is sometimes used with the 
same meaning as humus but in fact should refer to the sum of soil organic 
materials. 

Humified matter represents more than half soil total organic carbon 
and can be classified in two main types, humic and non-humic substances 
(Schnitzer 1978). The physical and chemical characteristics of non-humic 
substances e.g. carbohydrates, proteins, peptides, amino acids, lipids, 
waxes and organic acids of low molecular weight are easily recognizable. 

Humic substances on the other hand, do not show such marked 
physicochemical characteristics. They are more or less dark in colour, 
their molecular weight varies from a few hundred to several hundred 
thousand daltons, and they display a complex chemical structure, a 
hydrophilic character and acid properties. 



372 



Organic Analysis 



However, the distinction between the two types of substances is not 
completely clear since humic substances always contain non-humic 
substances, which can be released by chemical treatments like acid 
hydrolysis. 



cf. Chap.9 



Known 

molecular 

structures 



Oden 



Berzelius 



Organic 
residues 



Debris 



Fulvic 
acids 




Non-humic 
compounds 



Humic 
compounds 



Crenic 
acid 



Humic 
acids 



Apocrenic 
acid 



Brown 
humic acids 



Coloured 
polymers 



Berzelius 



Spring er 



Grey 
humic acids 



Light yellow Brown yellow Dark brown Dark grey 



Increase in degree of polymerization 

< 2,000 d Increase in molecular weight — ► 

45% Increase in carbon content — ► 

48% Decrease in oxygen content * 

1 ,400 Cmol(H + )kg" 1 Decrease in exchange acidity — ► 



> 300,000 d 

62% 

30% 
500 Cmol(H + )kg- 1 



Fig. 11.1. Classification and chemical properties of groups of humic substances 
(after Stevenson and Elliott 1989) 



Humic substances are generally classified into three main groups 
according to their solubility: 

- Humic acids are soluble in diluted bases and insoluble in acid medium; 
they result from precipitation by acidification of the alkaline extracts of 
soil. 

-Fulvic acids are soluble in both alkaline and acid mediums, the 
compounds remain in solution after precipitation of humic acids by 
acidification of the alkaline extracts of soil. 

- Humin is the humus fraction that cannot be extracted by acids or diluted 
bases. 



Quantification of Humic Compounds 373 

Other names have been given to certain humic substances as a function 
of their solubility in a range of different solvents. The best known 
characterize hymatomelanic acids as the fraction of humic acids which 
are soluble in ethanol. Among fulvic acids, an obsolete distinction is 
sometimes used to differentiate crenic and apocrenic acids. Some authors, 
for example Chamayou and Legros (1989), advise against the use of 
either term. Brown and grey humic acids can be distinguished. These two 
groups were first identified by Springer (1938) based on their flocculation 
or dissolution properties in a medium including different degrees of salts. 
Figure 11.1 shows some of the properties of these compounds. 

11.1.2 Role in the Soil and Environment 

Humic compounds account for 60-70% of soil carbon, which itself 
represents the biggest reservoir of organic carbon on the earth's surface. 
Humic compounds play a significant role as an atmospheric source of 
C0 2 and as a carbon reservoir which is likely to react to the influence of 
different external factors (Schnitzer 1978). 

Stevenson (1982) quoted nine properties of humus with respect to their 
effect on the soil: 

-Its dark colour, which facilitates absorption of solar radiation and 
consequently warms the soil. 

- Its water-retention capacity; organic matter can to hold up to 20 times 
its own weight in water, thereby significantly improving the hydrous 
properties of some soils and particularly of sandy soils. 

- Its ability to combine with clay minerals resulting in the cementing of 
soil particles into structural units called aggregates thereby facilitating 
gaseous exchange and increasing permeability. 

- Chelation which forms stable complexes with many polyvalent cations 
and influences the availability of nutriments for plants. 

- Its solubility in water which is very reduced and the bonds with clays 
and certain polyvalent cations which minimize organic losses by 
leaching. 

- Its buffer effect, this effect appears at slightly acid, neutral and alkaline 
pH. 

- Its cation exchange capacity, 20-70 % of the cation exchange capacity 
of many soils is due to the presence of organic matter. 

- Its mineralization which releases C0 2 , NH 4 + , N0 3 ~, P0 4 3_ , S0 4 2_ and 
represents a very significant source of nutrients for plant growth. 

- Its combination with other organic molecules which affects bioactivity, 
persistence and biodegradability of pesticides. 



374 Organic Analysis 

This chapter provides details of procedures for the main types of 
extraction and quantification of soil humic matter. The reference section 
at the end of the chapter lists a wider range of methods for use in this 
context. 

Extraction methods are described for whole soil samples prepared 
using standard techniques. However, it is often better to apply these 
methods to selected fractions after physical fractionation of the organic 
matter (cf. Chap. 9), in particular for the finest fractions. 

11.1.3 Extraction 

Organic materials bond with polyvalent cations, hydroxides and clays to 
form organomineral complexes. The stability of these complexes varies 
considerably depending on the type of bond. 

Organic materials can be released by extractions that break down at 
least some of the organomineral bonds. Bruckert (1979) distinguished 
three types of extraction solutions: 

- Salt solutions can break electrostatic bonds by simple exchange of ions 
and help solubilize the organic molecules by ionizing the acid and 
phenolic functional groups; Bruckert proposed a sodium tetraborate 
solution at pH 9.7; the extracted organomineral substances of relatively 
weak molecular weight were referred to as mobile or easily available 
recently formed complexes (Duchaufour 1977); they are characterized 
by relatively low metal contents; tetraborate has no effect on calcium 
complexes. 

- Complex-forming solutions able to break the coordination bonds; the 
best known is sodium pyrophosphate solution which is generally used 
at pH 9.8; it breaks the bonds of complexes with metal sites on clays; it 
can also solubilize complexes with high metal contents (amorphous 
hydroxides), and dissolve calcium humates by forming a complex with 
calcium, but is as ineffective as tetraborate on allophanic complexes; all 
the extracted complexes are known as immovable. 

- Soda at pH 12 is in fact the most effective extractant as it is able to 
destroy most organomineral bonds and particularly those of the humic 
allophone-acid complexes of andosols. 

The standard techniques described in Sect. 1 1.2.1 concern: 

- Simple extraction using an alkaline solution, with or without a 
preceding acid attack. 

- Double extraction using a pyrophosphate solution followed by a soda 
solution (this method is used in the IRD laboratories, France). 



Quantification of Humic Compounds 375 

Section 11.2 evaluates the respective utility and precision of these 
methods and describes the main methods for the purification of extracted 
organic materials. 

Some alternatives to these extraction methods, including a technique 
for fractionation of the humin centrifugation pellet, are presented in Sect. 
11.3. Techniques for the fractionation and characterization of humic 
compounds are described in Chap. 12. 



11.2. Main Techniques 



11.2.1 Extraction 



Principle 

As mentioned in Sect. 11.1.3, diluted soda solutions are the most 
powerful extracting reagents for humified matter. However, the use of 
these extraction solutions has been criticized for three main reasons 
(Bruckert 1979): 

- Neo-formation of soluble substances from non-humified plant 
materials. 

- Breakdown of humic substances by hydrolysis, oxidation or artificial 
polymerization. 

-Lysis of microbial organisms. Sodium hydroxide can destroy bacteria 

and empty them of their cytoplasmic contents, their cell walls then form 

a non-extractable residue. 

However, Schnitzer (1982) did not consider that extraction using 
diluted bases in a nitrogen atmosphere and at room temperature signifi- 
cantly modified the structure and characteristics of the extracted organic 
matter. Levesque and Schnitzer (1966) showed that 0.1 mol L" 1 soda 
solutions extract more organic matter than concentrated solutions. They 
also showed that 0.5 mol L" 1 soda solutions extract organic matter with 
lower ash content. 

"Method Schnitzer (1982)" and "Method IHSS" were chosen to maximize 
extraction of humic compounds and to minimize their degradation. 
Extraction (one extraction only) is carried out (1) under nitrogen 
atmosphere, with a 0.1 mol (NaOH) L" 1 soda solution described in 
"Method IHSS" (2) with the same reagent or 0.5 mol (NaOH) L" 1 soda 
solution or pyrophosphate solution described in "Method Schnitzer 
(1982)". "Method of Dabin (1976)" separates two types of extracted 



376 Organic Analysis 

compounds: organic materials extractable with a pyrophosphate solution 
at pH 9.8 and organic materials extractable later on with a 0.1 mol 
(NaOH) L" 1 soda solution. Negre et al. (1976) observed qualitative 
differences between the two extracts particularly in amino acid content; 
Thomann (1963) observed that pyrophosphate dissolves calcic humates 
by forming complexes with metal cations; an increase in pH acts more 
particularly on aggregate dispersion, and pH 9.8 corresponds to a stable 
stage in the curve of humus extraction as a function of pH. 

An alternative approach is acid pretreatment of the soil; this type of 
pretreatment facilitates the later extraction of humified matter by 
destroying carbonates and by solubilizing iron and aluminium 
hydroxides; however, the quantitative effect of the pretreatment is 
especially visible in calcareous soils. "Method IHSS" recommends 
systematic acid pretreatment with 1 mol (HO) L" 1 hydrochloric acid 
solution. "Method Schnitzer (1982)" recommends pretreatment with 0.05 
mol (H + ) L" 1 hydrochloric or sulphuric acid solution only in the case of 
calcareous soils. "Method of Dabin (1976)" recommends systematic 
pretreatment with 2 mol L" 1 phosphoric acid. This acid has two 
advantages (1) higher density (approximately 1.2) which is more 
favourable for the separation of light organic fragments (cf. Chap. 9), 
(2) it does not disturb wet carbon titration and thus enables quantification 
of the organic matter extracted by the acid itself (unbound fulvic acid). 

Equipment 

- Glass, polypropylene or polyvinyl extraction and centrifugation flasks 
(volume: 200 mL, and 300-500 mL) with screw caps, for use as 
centrifugation cylinders, capable of withstanding 10,000g. 

-Centrifuge (10,000g) equipped with rotor suitable for use with the 
centrifugation flasks. 

Products 

-Degassed inorganic water. Most commercial water is appropriate. It 
should first be checked for the absence of organic matter (blank assay 
corresponding to the type of characterization required). However, to 
eliminate organic matter from water, either (1) boil water for 2 h in the 
presence of 1% KMn0 4 and H 2 S0 4 then distil or (2) use deionized 
water purified on activated carbon (e.g. Millipore filter), then degas the 
water to eliminate dissolved oxygen in order to avoid oxidation of 
organic matter during extraction. Proceed either by boiling or by 
bubbling with nitrogen for 10 min. 



Quantification of Humic Compounds 377 

-0.1 mol (NaOH) L 1 solution. Dissolve 8 g of soda pellets in a 2 L 
volumetric flask in degassed inorganic water, complete to 2 L, agitate 
and store in a carefully stopped bottle. 

- 0.5 mol (NaOH) L 1 solution. Same as above with 40 g soda for 2 L. 

- 10 mol (NaOH) L 1 . Same as above with 400 g soda for 1 L. 

-0.1 mol (Na 4 P 2 7 ) L 1 solution. Dissolve 89.2 g of Na 4 P 2 O 7 ,10H 2 O in 

degassed inorganic water, complete to 2 L and store in a carefully 

stopped bottle. 
-0.1 mol (Na^ 2 7 ,NaOH)L ! solution. Dissolve 89.2 g of Na 4 P 2 O 7 ,10H 2 O 

and 8 g of soda pellets in degassed inorganic water, complete to 2 L and 

store in a carefully stopped bottle. 

- 2 mol (HCl) L 1 solution, dilute 166.7 mL of concentrated HC1 (d=\.\9) 
in degassed inorganic water; complete to 1 L. 

- 6 mol (HCl) L 1 solution. Dilute 500 mL of concentrated hydrochloric 
acid in 1 L degassed inorganic water. 

-1 mol (HCl) L 1 solution. Dilute 166.7 mL HCl in 2 L degassed 

inorganic water. 
-0.5 mol (HCl) L 1 solution. Dilute 83.3 mL HCl in 2 L degassed 

inorganic water. 
-0.05 mol (1/2H 2 S0 4 ) L 1 solution. Dilute 27.8 mL of concentrated 

H 2 S0 4 (d=1.81) in degassed inorganic water; cool and complete to 2 L. 
-2 mol (H^POj) L 1 solution. Dilute 136 mL of concentrated phosphoric 

acid (d=\.l\) in degassed inorganic water and complete to 1 L. 

Procedures 



Method Schnitzer (1982) 

If the soil contains carbonates (reaction to diluted hydrochloric acid), 
leave it in contact with a 0.05 mol (H + ) L" 1 hydrochloric or sulphuric acid 
solution at room temperature until the end of gaseous emission. Rinse the 
excess acid with inorganic water and leave the soil to dry on a plate at 
room temperature. 

Weigh 10 g of air-dried soil in a 200 mL polypropylene flask. Add 100 
mL of selected extraction solution (0.1 or 0.5 mol (NaOH) L" 1 , 0.1 
mol (Na 4 P 2 7 ) L" 1 or mix 0.1 mol (Na 4 P 2 7 ,NaOH) L" 1 . Purge the air out 
of the flask with a stream of nitrogen. Close carefully and agitate for 24 h 
at room temperature. Separate the dark supernatant solution from the 
solid phase by centrifugation (preferably for 10 min at 10,000g), suspend 
the residue in 50 mL degassed inorganic water, separate by centri- 
fugation again and add the flushing water to the previous solution. 



378 Organic Analysis 

Acidify the alkaline extract to pH 2 with 2 mol (HO) L 1 hydrochloric 
acid. Leave to stand for 24 h at room temperature then separate the 
soluble matter (fulvic acid) from the coagulated matter (humic acid) by 
centrifugation. The two fractions can be brought to dry by freeze-drying 
or by evaporation in a rotary evaporator at 40°C. 

Method IHSS 1 

Mix 20 g of air-dried soil with 1 mol (HO) L" 1 hydrochloric acid. Adjust 
to a pH of between 1 and 2 (15-20 mL soda 10 mol L" 1 ) in such a way 
that the final volume of liquid is 200 mL (liquid/soil ratio =10 mL per 1 
g). Agitate for 1 h and separate the supernatant liquid by centrifugation. 

Neutralize the centrifugation pellet to pH 7 with 1 mol (NaOH) L" 1 
soda solution and add 0.1 mol (NaOH) L" 1 solution under nitrogen 
atmosphere until a solution:soil ratio of 10:1 is obtained. 

Agitate for at least 4 h under nitrogen atmosphere. Leave to stand 
overnight and centrifuge. 

Acidify the centrifugation liquid to pH 1 with 6 mol (HO) L" 1 
hydrochloric acid under agitation. Leave to stand for 12-16 h and 
centrifuge to separate the fulvic acids in solution from the coagulated 
humic acids. 

Method of Dabin (1976) 

Put 40 g of air-dried soil crushed and sieved on a 0.5 mm mesh sieve in a 
300-500 mL centrifugation bottle. Add 200 mL of 2 mol (H 3 P0 4 ) L" 1 
solution, agitate for 30 min with the back and forth shaker and centrifuge 
for 5 min at l,500g. Filter the supernatant liquid on a flat filter in a 1 L 
glass bottle. Repeat the extraction two or three times in the same 
centrifugation bottle, filtering on the same filter and collecting the acid 
extracts in the same bottle. The filter contains light organic matter (LOM) 
from non-humified plant and animal residues (cf. Chap. 9); the acid 
solution contains a small fraction of organic materials called "free fulvic 
acids" (FFA) by Dabin (1976). Wash the centrifugation pellet two or 
three times with 200 mL inorganic water in the same bottle by agitating 
for 15 min; centrifuge and filter the washing water on the previously used 
filter to collect the light material still present in the washing water, 
discard the filtrate. 



IHSS = International Humic Substance Society, Univ. of California, Los Angeles, 
CA 90024; Federal Center, mall stop 407, Box 25046, Denver, CO 80225. 



Quantification of Humic Compounds 379 

Add 200 mL of 0.1 mol (Na 4 P 2 7 ) L" 1 solution at pH 9.8. Agitate for 4 
h on the back and forth shaker or leave in contact overnight agitating 
several times. Separate the supernatant liquid by centrifugation for 30 
min at 3,000g and transfer it through a filter into a 1 L volumetric flask. 
Perform a second extraction in the same conditions and combine the 
extracts. If the second extract is dark in colour, perform a third extraction. 

Repeat a similar extraction sequence on the centrifugation pellet, with 
0.1 mol (NaOH) L" 1 soda solution instead of the pH 9.8 pyrophosphate 
solution. 

Humic acids of the pyrophosphate and soda extracts are separated from 
fulvic acid by acidification at pH 1 with the 2 mol (HO) L" 1 solution as 
described in "Method Schnitzer (1982)". Ultimately, the following 
fractions are obtained: LOM, FFA, pyrophosphate fulvic acids (PFA), 
pyrophosphate humic acids (PHA), soda fulvic acids (SFA), soda humic 
acids (SHA), extraction residue or humin. 

Note 

In certain soils, the pyrophosphate and soda extracts can contain a high 
percentage of fine mineralogical clays (smaller than 0.2 |um). It is 
possible to separate these clays by flocculation with the addition of a little 
potassium sulphate, but there is a risk of simultaneously flocculating 
certain grey humic acids (cf. Sect. 11.2.4 and Chap. 12). After 
centrifugation, the flocculation pellet should either be titrated 
individually or combined with the previous humin pellet for carbon 
determination. 



11.2.2 Quantification of the Extracts 



Principle 

Quantification is by carbon titration of each extract (cf. Sect. 11.2.1 
above). The techniques used for carbon titration of whole soil can be used 
on the humin pellet (cf. Chap. 10). For LOM, it is preferable to use a 
combustion technique. The extracts can also be titrated by combustion on 
the residue after dry evaporation of an aliquot. Nitrogen can be measured 
in addition to carbon, hydrogen and possibly sulphur and oxygen by 
using an analyser of the CHN type. However, wet processes such 
as dichromate oxidation (described later) are often preferred for 



380 Organic Analysis 

titration of the extracts. Another technique calls for a titration apparatus 
with dissolved carbon. Many of these apparatus are based on titration of 
the carbon dioxide (generally by infra-red absorption) produced by 
oxidation of the solution with a powerful oxidant. The titration apparatus 
of dissolved carbon are rather expensive and reserved to precise 
environmental studies; the manufacturer's instructions should be 
respected. 

In acid medium, dichromate oxidizes the carbon of the organic matter 
in C0 2 according to the redox reaction: 

Cr 2 7 2 - + 6 e- + 14 H + -> 2 Cr 3+ + 7 H 2 
C + 2 H 2 -> C0 2 + 4e- +4 H + 

With automated apparatuses, the quantity of C0 2 released can be 
measured directly. With a traditional manual redox technique a 
dichromate excess is used, which is then back titrated by a ferrous iron 
solution: 

Fe 2+ -> Fe 3+ + e - 

A mole of ferrous iron corresponds to 1/6 mol of K 2 Cr 2 7 is 1/4 atom 
C or 3 g of carbon. 

Equipment 

- 50 and 100 mL Pyrex Beakers. 

- Precision burette (25 or 50 mL). 

- If necessary, a titration apparatus using carbon and dry combustion, but 
preferably a wet process. 

Reagents 

- 0.1 mol (Na 4 P 2 7 ) L _1 and 0.1 mol (NaOH) L 1 solutions: see 
preparation in "Products" under Sect. 11.2.1. 

- Concentrated sulphuric acid (J=1.81). 

- 2 mol ( 1 /2H 2 S0 4 ) L _1 sulphuric acid: dissolve 56 mL of concentrated 
sulphuric acid (J=1.81) in 1 L inorganic water. 

- 0.1 mol ( 1 /2H 2 S0 4 ) L _1 sulphuric acid: 2.8 mL of concentrated sulphuric 
acid (J=1.81) in 1 L inorganic water. 

-2% potassium dichromate solution: dissolve 20g of K 2 Cr 2 7 in 
approximately 400 mL inorganic water, slowly add 500 mL of 
concentrated sulphuric acid, agitate, let cool and complete to 1 L with 
inorganic water. 

- 0.5 mol (y6K 2 Cr 2 7 ) L _1 solution: gradually dissolve 24.52 g of 
potassium dichromate in inorganic water then complete to 1 L (solution 
for the titration of the Mohr salt). 



Quantification of Humic Compounds 381 

- 0.2 mol (FeS0 4 ) L _1 Mohr salt solution: dissolve 78.4 g of Mohr salt 
(FeS0 4 ,(NH 4 ) 2 S0 4 ,6H 2 0) in 500 mL water, add 20 mL of concentrated 
sulphuric acid, complete to 1 L. 

- Sodium fluoride in powder form. 

- Sulphuric diphenylamine solution: dissolve 0.5 g of diphenylamine 
powder in 100 mL of concentrated H 2 S0 4 , pour into 20 mL water, 
agitate and store in a brown bottle. 

Procedure 



Test samples 

Total organic materials of the soda and pyrophosphate extracts. In a 
100-200 mL shallow beaker, put the exact volume of the extraction 
solution corresponding to 5-8 mg C. Calculate the volume of the aliquot 
on the basis of the total C analysis of the sample (cf Chap. 10). The 
carbon of the total alkaline extracts is around 40% of total C; C extracted 
by pyrophosphate is about 25% of total C, and C extracted by soda (after 
pyrophosphate extraction) is about 15% of total C. Before titration, bring 
the sample to dry in a drying oven at 70°C. 

Humic acids. Take a sample of the extraction solution of more than 
50% of the titration sample for total organic materials. Precipitate the 
humic acids at approximately pH 1 by adding 1 mol (H 2 S0 4 ) L 1 
(approximately 4-5 mL for 10 mL of total extract, 3 mL for 10 mL of 
pyrophosphate extract, 1.5 mL for 10 mL of soda extract); leave to 
flocculate for at least 4 h and centrifuge for at least 5 min at 3,500g; 
separate the supernatant liquid (fulvic acids) and wash with 0.1 
mol (!/2H 2 S0 4 ) L _1 solution. Dissolve the centrifugation pellet in 0.1 
mol (NaOH) L" 1 solution, place in a beaker and bring to dry at 70°C 
before analysis. 

Phosphoric acid extract. Take an exact aliquot of 100 mL; concentrate 
in the drying oven until approximately 10 mL remains (H 3 P0 4 cannot go 
dry) and carry out titration on this concentrated solution. 

Fulvic acids. These can be titrated as total organic material after 
elimination of humic acids but are generally estimated by the difference 
between total organic materials and humic acids. 

Redox Titration 

After drying the sample specimens in the beakers, add 10 mL of the 2% 
potassium dichromate solution in sulphuric acid medium. At the same 
time, carry out a blank measurement with 10 mL of the same dichromate 



382 Organic Analysis 

solution in a beaker. Protect each beaker with a beaker cover and bring to 
a very gentle boil on a hotplate regulated at 215-220°C; Boil for 5 min 
but control boiling to avoid overheating or too much evaporation. 

Leave to cool, rinse the beaker cover and add in the beaker: 100 mL of 
inorganic water, 1.5 g of NaF (or 2.5 mL H 3 P0 4 ) and three drops of 
diphenylamine solution. 

With a burette titrate with Mohr salt solution until the colour turns 
from purple to pale green. V and V volumes in mL of ferrous solutions 
are necessary for the respective titration of the blank and of the sample. 

If T is the concentration of the Mohr salt solution in mol (FeS0 4 ) L" 1 , 
the quantity of Fe 2+ equivalent to oxidant mobilized for the titration of the 
carbon of the sample is: T(V-V) mmol(Fe 2+ ), which according to the redox 
equations (cf "Principle" under Sect. 11.2.2), corresponds to 3T (V -V) 
mg of carbon in the beaker. With respect to the whole soil sample, the 
carbon content in mg (C) g" 1 (soil) of the fraction is 

C = 3T(V-V) V t /(Am t ) (11.1) 

where: 

V t \ total volume of the humic extract in mL, 
A: aliquot of the humic extract used for titration in mL, 
m t \ soil sample mass used for the extraction in g, 
T has to be determined by titration. 

Titration of the Mohr Salt Solution 

Put in a 250 mL beaker: 

- exactly 10 mL of the 0.5 mol (1/6 K 2 Cr 2 7 ) L" 1 solution 

- 100 mL of inorganic water 

- 15 mL of concentrated H 2 S0 4 
Let cool and add 

- 3.75 g NaF; 

- three drops of diphenylamine indicator 

Using the burette, titrate with the Mohr salt solution; if Vf is the 
volume in mL of the Mohr salt solution, the T of this solution will be 

T = 5/Vf mol (Fe 2+ ) L 1 . The carbon content in (11.1) can thus be 
expressed in mg (C) g" 1 (soil) by: 

C = 15 (V-V) V t /(A m t V f ) (11.1') 



Quantification of Humic Compounds 383 

Remarks 

Ten millilitres of 2% dichromate solution corresponds to 20.4 mL of the 
0.2 mol (Fe 2+ ) L _1 Mohr salt solution. The volume of Mohr salt solution 
used for titration of the sample should be between 7 and 15 mL; below 
this range, start the analysis again with a weaker sample; above this range 
start again with a stronger sample. 

The quantity of carbon in the phosphoric acid solution is usually low. 
In this case, oxidize with only 5 mL of 2% dichromate solution adding 
five drops of concentrated H 2 S0 4 before boiling. 

Pyrex beakers are attacked by alkaline solutions and NaF in acid 
medium. They should be rinsed immediately after titration and reserved 
solely for this purpose. 



11.2.3 Precision and Correspondence of the Extraction Methods 

Inter-Laboratory Study 

An inter-laboratory study by GEMOS 2 (Dabin et al. 1983) involved the 
quantitative comparison of the quantities of organic materials extracted 
on seven samples from soils from different areas of France: 

1. A silt soil from the plates of Boigneville 

2. A humocalcic soil from Pontarlier 

3. A Al horizon of a podzol from the forest of Villers Cotterets 

4. A Bh horizon of the same podzol 3 

5. A fersiallitic soil from near Montpellier 

6. A gley soil with hydromull from Bonneveaux 

7. A rendzina on chalk from Chalons sur Marne 

Each soil was analysed by four French laboratories: 

- the CIRAD 3 laboratory of Montpellier 

- the pedology laboratory of the university of Poitiers 

- the IRD 4 laboratory of Bondy 

- the pedology laboratory of the university of Besancon. 



2 GEMOS = Groupe d'Etude des Matieres Organiques des Sols, sub-group of the 

Association Frangaise d'Etude des Sols (AFES), INRA, 78850 Thiverval- 
Grignon, France. 

3 CIRAD = Centre International de Recherche Agronomique pour le 

Developpement, Avenue d'Agropolis, BP 5035, 34032 Montpellier Cedex, 
France. 

4 IRD = Institute of Research for the Development (ex-Orstom), 32 Avenue 

Varagnat, 93143 Bondy, France. 



384 



Organic Analysis 



The method of reference chosen for the comparisons was described in 
"Method IHSS". In addition, the IRD laboratory in Bondy tested the method 
described in "Method of Dabin (1976)" on the same samples. 

Quantities Extracted by the IHSS Method 

The results of the inter-laboratory study are summarized in Fig. 11.2. 
Figure 11.2a shows the carbon value in g for 100 g of dry soil obtained 
by the sum of the three fractions: acid extract + alkaline extract + non- 
extracted residue, compared to total carbon measured on whole soil. The 
fact that the results are located close to the bisecting line shows the 
absence of bias between the two methods. 



C-sum effractions % 
10 




10 
total-C % 



C-alkaline extract % 


6 - 


♦ 


* 


4 - 


- i^l 




^^^ ♦ 


2 - 


^#^ 




^k X X 





^* 


i i i i i i i i ii 


5 10 


C-sum effractions % 



C-acid extract % 


0,8 \ 


♦ 


~ 


0,6 \ 




i/ 


0,4 : 


j^\. 


^r 


0,2 : 
n 


J^ o * 


U i i -p 

5 10 


C-sum effractions % 




2 4 6 8 

C-alkaline extract % 



Fig. 11.2. Results of an inter-laboratory comparison using the extraction method 
described in "Method IHSS" (unpublished data). Results from four 
laboratories for the seven types of soils described in the text: (plus) soil 
1, (open triangle) soil 2, (open square) soil 3, (open diamond) soil 4, 
(cross) soil 5, (filled diamond) soil 6, (open circle) soil 7 



Quantification of Humic Compounds 385 

Figure 11.2b shows the amount of carbon extracted with the 
hydrochloric acid solution compared to the total quantity extracted. The 
quantity of acid extracted was low, below or equal to 5% of total carbon. 
The two exceptions where the value was 10% are probably due to the 
presence of additional fragments of light non-humified organic matter. 
The values measured were very variable and there was no correlation 
with total carbon. 

Figure 1 1.2c shows the carbon of the alkaline extract compared to total 
carbon. Two remarks can be made: 

-in six of the seven soils, the soda solution extracted 20-40% of total 

carbon. The values are more grouped than that of the acid extract. Their 

distribution revealed a maximum threshold of extraction for six samples 

located at approximately 40% of total carbon (line). The values located 

under this threshold are probably errors in measurement due to 

insufficient extraction. 

- there was an abnormally high value for soil 4; this can be explained by 

the fact the soil was from the deep Bh organic horizon of a podzol; the 

organic matter which was leached to this depth was much more soluble 

in the soda solution than in the six other soils; in this case, the reagent 

extracted more than 3/4 of the carbon of the sample. 

Figure 11. 2d shows the quantity of carbon in humic acid forms 

compared to the carbon of the total alkaline extract. Whatever the type of 

soil, the carbon of humic acids accounted for approximately the 2/3 of the 

carbon of the alkaline extracts, and thus approximately 27% of total 

carbon. The deviation of the results around this value was limited. 

Precision of the IHSS Method 

Table 11.1 shows the results of an analysis of variance carried out on the 
data obtained with the IHSS method: are the measured values on each soil 
equal or significantly different compared to experimental error. 

The F test is the ratio of variance between soils (seven soils i.e. six 
degrees of freedom dof) to within inter-laboratory variance (27 
measurements or 20 dof) represented by s r 2 . The pooled estimation of the 
standard error associated with a measurement from a given laboratory on 
an unspecified soil is indicated by "s" in g (C) 100 g" 1 (dry soil) in 
absolute values and by RSD (relative standard deviation) in relative 
values. These values representing the precision of an inter-laboratory 
reproducibility test are upper limits of error. Repeatability would be 
better within one laboratory with well-trained staff. 



386 Organic Analysis 

Carbon measurement was tested in the hydrochloric acid extract, in the 
soda extract, in the humin residue, in the sum of these three fractions and 
finally in humic acids and fulvic acids. 

Table 11.1 shows that: 

- It is impossible to control the quantities extracted by hydrochloric acid, 
the errors observed in this case being more significant than the variations 
between the soils; this confirms the distribution in Fig. 1 1.2b. 

- In all the other cases, the differences observed between the soils were 
significant compared to the residual error representing inter-laboratory 
variability. 

Table 11.1. Precision of measurements in a comparative inter-laboratory test on 
seven soils using the IHSS method (F: test of significance of the soil 
values compared to the residual variance s 2 r between the four 
laboratories (***, significant difference between laboratory data at 
risk <1 %, NS, no significant difference), s and RSD; expected 
absolute (g (C) 100 g~ 1 dry soil) and relative (%) standard deviations in 
case of a measurement (no replicate) from any laboratory) 



Determination 


F(6,21) 


S 2 r 


s 


RSD% 


C- acid extract 


4.2 (NS) 


0.026 


0.16 


83 


C- alcaline extract 


107 *** 


0.141 


0.38 


17 


C- humin residue 


37 *** 


0.317 


0.56 


22 


C- sum of fractions 


83 *** 


0.410 


0.64 


13 


C- total soil 


100 *** 


0.403 


0.63 


13 


C- humic acids 


67 *** 


0.131 


0.36 


22 


C- fulvic acids 


16 *** 


0.036 


0.19 


35 



- In the alkaline extracts, the precision of the measurement of humic 
acids is better than that in the fulvic acids; this is probably due to the 
greater abundance of humic acids. 

- The precision of the measurement of soil total carbon obtained by the 
sum of the first three measurements is of the same order of magnitude 
as that obtained by the direct measurement of carbon on the whole soil; 
moreover, the value obtained is in agreement with the rule of 
propagation of errors (Pansu et al. 2001); indeed, C-sum is obtained by: 
C-sum = C-acid extract + C-alkaline extract + C-humin residue 



Quantification of Humic Compounds 



387 



In the case of normal laws: 

s C-sum = ( s C-acid extract + s C-alcaline extract + s C-humin) 
then: 

s C-sum = (°- 026 + °- 141 + 0.317) 1/2 

s C-sum = 0-^9 is very close to the values 0.64 and 0.63 found for the 

synthetic variable and the measurement of total carbon, respectively. 

Comparison of the Methods Described in "Method IHSS" and 
"Method ofDabin (1976)" 



12 
10 
8 
6 
4 
2 




C2 = C-sum of fractions of method of Dabin 



C2=(1.01 +0.05)C1 
^ = 0.99 




2 4 6 8 10 12 

C1 = C-sum of fractions of method IHSS 



Fig. 11.3. Comparison of the carbon rates found with the two methods of 
extraction tested. Averages of the results of four laboratories for the 
seven types of soils described in the text: (plus) soil 1 , (open triangle) 
soil 2, (open square) soil 3, (open diamond) soil 4, (cross) soil 5, 
(filled diamond) soil 6, (open circle) soil 7 

Figure 11.3 shows that the total quantities of carbon (acid extract + 
alkaline extract + humin residue) found with the two methods are very 
close. However, more detailed comparison of the data in Fig. 1 1.4 reveals 
analogies and differences between the methods: 

-Extraction of phosphoric acid described in "Method ofDabin (1976)" 
solubilizes between 1.2 and 4 times more (average 2 times more) 
organic matter than extraction with hydrochloric acid described in 
"Method IHSS" (Fig. 11.4a); the closest results for the two techniques 
were for the deep Bh horizon of the podzol. 
- Alkaline extraction gave quantitatively comparable results with the two 
methods (Fig. 1 1 .4b) when the sum of the extracts pyrophosphate and 



388 



Organic Analysis 



soda described in "Method of Dabin (1976)" are taken into account; the 
results are also comparable for humin carbon, although very slightly 
weaker in the method described in "Method of Dabin (1976)", (Fig. 
11. 4d). 



"Method of Dabin C-acid extracts % 

(1976)" 
1 — I — 




1 — i — I — [ill 1 — I — | — i — < — r 



0,2 0,4 0,6 0,8 

"Method IHSS* 




2 3 4 

'Method IHSS" 



Method of Dabm c-alkaline extracts %| 
(1976)' ' ^ 

6 




12 3 4 5 6 

"Method IHSS" 



"Method of Dabin 
(1976)" 

6 a 

5 \ 

4 :- 

3 j- 

2 : 

1 -.- yl 

1 " ■ I ■ ■ ■ ■ I ■ 



C-humin residue % 



y 



y 



S x 



12 3 4 5 

"Method IHSS" 



Fig. 11.4. Comparison of the fractions extracted with the two extraction methods 
in the comparative study. Averages of the results of four laboratories 
for the seven types of soils described in the text: (plus) soil 1 , (open 
triangle) soil 2, (filled triangle) soil 3, (open diamond) soil 4, (cross) soil 
5, (filled diamond) soil 6, (open circle) soil 7. 



On the other hand, there was a clear difference between the two 
methods with respect to the quality of the extracted organic matter since 
"Method of Dabin (1976)" provided significantly less humic acids; in 
Fig. 1 1.4c the C- "Method of Dabin (1976)"/C- "Method IHSS" ratio is 
approximately 2:3 and the behaviour of sample 4 (deep Bh horizon of 



Quantification of Humic Compounds 389 

the podzol) is a little different; the difference may be due to the effect 
of the extracting reagent on the extracted molecules i.e. polymerization 
in "Method IHSS", macromolecular breakdown in "Method of Dab in 
(1976)" (cf. remarks in the introduction to this chapter). 

11.2.4 Purification of humic Materials 



Introduction 

It is difficult to choose a precise procedure for purification, as many 
alternatives are possible. According to Schnitzer (1982), the prime 
objective of purification is to minimize the weight of ash, while the 
second objective consists in separating the organic molecules of lower 
molecular weight from the humic materials. However, these definitions 
may be insufficient because the modes of the bonds between humic, non- 
humic and inorganic extracted materials are very complex and it is 
possible that many methods of purification have an influence on the 
structure of the final compounds isolated. 

Negre et al. (1976) recommend dialysis (Viskins dialysis bags with 24 
A pores) to purify humic materials extracted by pyrophosphate. Like 
other authors before them, these authors noted some transformation of the 
fulvic acids into humic polymers of higher molecular weight. It is as if 
"during the dialysis, which is accompanied by a progressive return of the 
medium towards neutrality, the molecules that were depolymerized 
during the alkaline extraction could polymerize again by simple re- 
establishment of the CO-NH bonds, similar to the peptide bonds leading 
to the formation of the nucleic acids" (Negre et al. 1976). 

Other simple purification methods enable elimination of certain 
minerals from the extracted solution by simple coagulation with the 
addition of a little sodium sulphate and centrifugation (Kumada et al. 
1967) or ultracentrifugation (Jacquin et al. 1970). 

Humic acids can also be purified by dissolving them in soda medium 
then precipitating them again in acid medium (Lowe 1980) or by a 
second process of extraction-fractionation on extracts that have pre- 
viously been freeze-dried (Schnitzer 1982), or by prolonged freezing of 
humic solutions which can fractionate the different phases (Bachelier 1983). 

The most effective procedure for purification - though only of humic 
acids - is to chemically attack the minerals with a diluted solution of the 
HC1-HF mixture to reduce the weight of ashes. Schnitzer (1982) noted 
that HC1-HF treatment can reduce the ash contents to less than 1%. 



390 



Organic Analysis 



Jacquin et al. (1970) obtained less than 3% of ashes on three extracts 
purified with HC1-HF. Among the four purification methods tested by 
these authors, infra-red absorption spectra of purified humic materials 
showed that only the HC1-HF treatment almost completely eliminates the 
intense absorption bands of the phyllosilicates at 470, 520 and 1,030 
cm" 1 (Fig. 11.5). However, according to these authors, the hydrofluoric 
treatment leads to transformation of the chemical structure of the 
molecules, in particular a significant reduction in carboxylic acidity. 




4000 



3000 



2000 1 600 



1000 



300 



cm 



Fig. 11.5. Study by IR absorption spectrometry of the effect of three methods of 
purification on humic acids extracted from a deep Bh horizon of podzol 
(Jacquin et al. 1970): 1 , non-purified humic acids; 2, humic acids purified 
by percolation on OH~ and H + resins; 3, humic acids purified by ultra- 
centrifugation; 4, humic acids purified by HCI-HF treatment 



Quantification of Humic Compounds 391 

Fulvic acids can be purified in acid medium by adsorption on non- 
ionic standard polyacrylic resins of the amberlite XAD-7 type (Aiken 
et al. 1979). After rinsing the resin in slightly acid medium to eliminate 
mineral salts, more than 98% of the fulvic acids can be recovered by 
elution at pH 6.5 (Gregor and Powell 1986). A simpler solution consists 
in eliminating the metal cations by repeated exchanges on a cation 
exchange resin in H + form (Schnitzer 1982). 

Equipment 

- 150 mL Teflon or polypropylene bottles 

- Glass columns for chromatography with a diameter of 1 or 2 cm and a 
Teflon stopcock 

- Optional. Freeze dryer and freezer at -18°C 

Products 

- Dialysis bags with 24 A pores 

- Non-ionic polyacrylic resin (Amberlite XAD-7 or similar) 

- Cation exchange resin Amberlite IR 120, or Dowex 50, in H + form 

- HCl-HF mixture. Dilute 5 mL of concentrated HC1 and 5 mL of 52% 
hydrofluoric acid in 990 mL inorganic water 

- Extracting reagents, see Sect. 11.2.1 

Procedures 

Only the techniques for the purification of fulvic acids by cation 
exchange resins and techniques of purification of humic acids by HCl-HF 
attack are described here. However, as mentioned in "Introduction", it is 
important to be very careful when choosing a purification method, which 
should take into account observations that need to be carried out on the 
purified products later on. For other methods of purification, see 
references in the last paragraph of "Introduction" under Sect. 1 1.2.4. 

Agitate a mixture of 1 g of humic acids and 100 mL of HCl-HF 
solution in a stopped polypropylene flask for 24 h at room temperature. 
Filter and suspend the filtrate again with 100 mL of HCl-HF solution. 
Repeat this treatment three or four times, carefully rinse the residue with 
inorganic water, then dry or freeze-dry. 

Purify the fulvic acid solution three or four times on cation ex-change 
resin in H + form, then freeze-dry. 



392 Organic Analysis 

11.3. Further Alternatives and Complementary Methods 



11.3.1. Alternative Methods of Extraction 

Though the methods described above using diluted soda and sodium 
pyrophosphate are the most widely used, alternative methods of 
extraction have been proposed. For example, Kumada et al. (1967) 
developed a method used at the University of Nagoya (Japan); Lowe 
(1980) used yet another technique at the University of British Columbia 
(Canada); and the two techniques were the subject of a comparative test 
by Lowe and Kumada (1984). 

Gregor and Powell (1986) developed a method for the extraction of 
fulvic acids with pyrophosphate in acid medium; this technique could 
avoids two potential problems: oxidation of the phenolic compounds in 
alkaline medium, and oxidation by the Fe 3+ ions during acidification for 
precipitation of humic acids. 

Several comparative studies of extractions should also be mentioned. 
For example, Thomann (1963) compared 3% ammonium oxalate, 1% 
soda, 1% sodium fluoride and sodium pyrophosphate; Jacquin et al. 
(1970) compared soda, sodium pyrophosphate and ion exchange resins; 
Hayes et al. (1975) compared saline solutions, organic chelating agents, 
dipolar aprotic solvents, pyridine, ethylene diamine and soda in solution. 
They concluded that soda is the best of the reagents they tested for 
isolation of representative extracts of a broad range of humic substances. 

11.3.2 Fractionation of the Humin Residue 



Principle 

Given that between 40 and 80% of total carbon cannot be extracted with 
alkaline solvents, Perraud (1971) and Perraud et al. (1971) proposed a 
technique for the fractionation of the non-extractable humin residue. 
They performed successively: 

- An alkaline extraction after two attacks with hot H 2 S0 4 : this extract 
was called "humin bound to iron" (Perraud et al. 1971); the sulphuric 
attack probably also releases sugars during the destruction of non- 
extractable polysaccharide like cellulose (cf Chap. 13). 
-An alkaline extraction after six attacks with hot HF-HC1 provided 
organic material bound to clays. 



Quantification of Humic Compounds 393 

However, these authors showed that the non-extractable fraction was 
still very high (37-52% of total carbon in their test) in spite of the total 
destruction of clays which was confirmed by X-ray. From 8 to 23% 
of this non-extractable fraction solubilized in CH 3 COBr probably 
corresponded to fresh or only slightly transformed organic matter that 
was not previously trapped in a mineral gangue. The fraction that 
remained insoluble in CH 3 COBr probably corresponded to either (1) 
organic matter very near to lignin but sufficiently transformed to be 
insoluble in acetyl bromide or (2) highly polymerized compounds in 
which the reduction of the functional groups probably resulted in their 
becoming insoluble in alkaline reagents. 

A procedure for fractionation of the humin residue developed from the 
method of Perraud et al. (1971) and used in IRD laboratories (Bondy, 
France) is described below. 

Procedure 

- Weigh 10 g of the extraction residue of Sect. 1 1.2.1 above that has been 
dried, crushed and sieved to 0.2 mm. 

- Add 50 mL of 1 mol ( 1 /2H 2 so 4 ) L" 1 sulphuric acid and heat at 70°C for 
3h. 

- Centrifuge at 3,000g for 15 min. 

- Wash twice with hot water and recover the centrifugation pellet. 

- Extract the centrifugation pellet with 50 mL 0.1 mol (NaOH) L" 1 soda 
solution for 4 h with agitation and leave to stand overnight. 

- Centrifuge at 3,000g for 15 min. The supernatant liquid contains humin 
bound to hydroxides (HH); reserve for titration of fulvic and humic 
acids as described in Sect. 11.2.2; depending on soil type, the extract 
can be purified by flocculation of clays with a salt like sodium sulphate; 
add the flocculate to the centrifugation pellet. 

- Take an aliquot of the centrifugation pellet for intermediate titration of 
carbon and possibly of nitrogen; this titration enables identification of 
the fraction that is solubilized in the acid (e.g. polysaccharides) by 
calculating the difference. 

- On the other fraction of the centrifugation pellet (the larger fraction), 
destroy the clay particles by: 

(a) four successive attacks with 50 mL of the 1 mol (HC1-HF) L" 1 
mixture at 70°C for 3 h then centrifuge for 15 min at 3,000g 

(b) Attack with 50 mL of the 1 mol (HF) L" 1 solution for 3 h at 70°C, 
centrifuge at 3,000g for 15 min, wash the centrifugation pellet with hot 
water and centrifuge again. 



394 Organic Analysis 

-Extract the centrifugation pellet with 50 mL of 0.1 mol (NaOH) L" 1 

solution agitate for 4 h and leave to stand overnight. 
-Centrifuge at 3,000g for 15 min. Recover the humic compounds of 

humin bound to the silicates (HS) from the solution; on these 

compounds measure the carbon of the fulvic and humic acids (cf. Sect. 

11.2.2). 
-Inherited humin(IH), which is made up of small organic fragments 

resembling charcoal, can be recovered on the centrifugation pellet: 

(a) Add 50 mL H 3 P0 4 d = 1.4; subject to ultrasound for 10 min, agitate 
mechanically for 30 min; 

(b) Centrifuge at l,500g for 10 min, then filter the supernatant on a 
small funnel stopped with a glass wool plug. 

The carbon fragments recovered on this plug are the insoluble IH. As 
the liquids of previous acid attacks can also include suspended particles, 
after each centrifugation it is advised to filter the supernatants on the 
same glass wool plugs. After careful rinsing with inorganic water, dry the 
funnel at 50°C, crush the inherited humin and glass wool plug with an 
agate mortar and analyse carbon and nitrogen with a CHN analyser. 

The last centrifugation pellet is the final residue of nonextr actable 
residual humin (RH), rinse twice with inorganic water to eliminate the 
phosphoric acid, dry, crush and analyse carbon and nitrogen with a CHN 
analyser. 

Calculations 

Data Collected 

Weight of soil sample at the beginning: P0 

Weight of humin (centrifugation pellet cf. "Procedures" 

under Sect. 11.2.1): PI 

Weight of the PI sampling for sulphuric attack: P2 

Weight of the intermediate pellet (after HH extract): P3 

Weight of the P3 sampling for HF-HC1 attack: PA 

Possible weight of other P3 sampling for C titration: P '4 

(generally, P4 + P4' = P3) 

Weight of non-extractable RH: P5 

Concentrations of the Extracts 

Humin (initial centrifugation pellet cf. "Procedures" 

under Sect. 11.2.1) Ch 

Humin bound to hydroxides (mg C on initial humin) Chh 

Intermediate centrifugation pellet (after HH extract) mg C g" 1 Ci 



Quantification of Humic Compounds 395 

Humin bound to silicates (mg C on the whole extracted) Chs 
Carbon from the mixture of the glass wool 

and inherited humin (mg) Cih 

Residual humin (mg C g 1 ) Crh 

Calculations 

Calculated on the initial soil sample, the carbon concentrations, (mg C 
g 1 dry soil) of humin before fractionation (H), humin bound to the 
hydroxides (HH), intermediate residue (I), humin bound to silicates (HS), 
inherited humin (IH), residual humin (RH) are expressed by: 

U = ChPl/P0 

HH = Chh P\ (P2 PO)- 1 

I = CiP3Pl (P4'P0)~ l 

HS = Chs P3 PI (PA P2 PO) 1 

IH = Cih P3 PI (PA P2 PO) 1 

RH = Crh P5 P3 P\ (PA P2 PO) 1 

The H-(I+HH) value provides an estimate of the carbon dissolved in 
the hot sulphuric acid (polysaccharides of humin). The I-(HS+IH+RH) 
value provides an estimate of the carbon dissolved by the HF-HC1 
mixture. 

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Thomann Ch (1963) Quelques observations sur l'extraction de l'humus dans les 

sols : methode au pyrophosphate de sodium. Cah. ORSTOM ser. 

PedoL, 3, 43-72 



12 



Characterization of Humic Compounds 



12.1 Introduction 



12.1.1 Mechanisms of Formation 

The synthesis of humic substances has been the object of speculation for 
many years. Andreux (1994) distinguished lytic mechanisms (lysis of the 
cellular walls, proteolysis, ligninolysis, transformation of polyphenols 
and other organic components), the mechanisms of tanning and melanisation 
which include incorporation of nitrogen and oxygen (Maillard's reaction: 
condensation of carbohydrates in the presence of amino nitrogen, 
condensation of polyphenol and amino acids in oxidizing medium) and 
the incorporation of inherited compounds. 

Schnitzer (1978) reported the following four hypotheses about the 
formation of humic substances: 

- Deterioration of plant material. Certain fractions of plant tissues, 
particularly woody materials, are only superficially decomposed in the 
soil to form humic substances; the nature of this "inherited humus" is 
thus strongly influenced by the nature of the original plant material; the 
first stage of humiflcation provides the heaviest humic substances 
which can then be broken down into lighter substances and ultimately 
into C0 2 and H 2 0. 

- Chemical polymerization. The plant materials break down into small 
molecules which are used as a source of energy and carbon by micro- 
organisms; these micro-organisms then synthesize phenols and amino 
acids which are polymerized into humic substances; in this case the 
nature of original material has no effect on the type of substance 
formed. 



400 



Organic Analysis 



- Cellular autolysis. The fragments resulting from autolysis of microbial 
and plant cells (amino sugars, acids, phenols and others aromatic 
compounds) condense and polymerize via free radicals. 

- Microbial synthesis. Microbes use plant tissue as a source of carbon and 
energy to synthesize intercellular organic materials of high molecular 
weight; at microbial death, these substances are released in the soil; 
they represent the first stage of humification and can then undergo 
extracellular microbial degradation into lighter molecules. 

12.1.2 Molecular Structure 

Based on their solubility properties, humic substances are generally 
classified in the three following categories: humic acids, fulvic acids, 
humins (cf. Chap. 11). 



C7Jj A ! j 





Fig. 12.1. Structure of two humic macromolecules; only the nuclei are 
represented at a scale of molecular weight (mw) for (a) mw >50,000, 
and (b) mw <5,000. filled circle C=0, open circle CH, open square NH, 
— peptide bond, • — OH carboxyl (Andreux, 1994) 



In a review of the literature, Schnitzer and Kahn (1972) showed that 
these three humic fractions are structurally rather similar. The structure of 
the humic molecule can be described schematically as a nucleus rich in 
hydroxy-quinonic units linked by C-C or C-O-C bonds (Andreux 1994). 
The frequency of distribution of the proteic and polypeptide chains fixed 



Characterization of bfrnic Compounds 401 

on this nucleus varies with the type of soil and the precursors. The 
molecular size of these structures (Fig. 12.1) depends both on the 
dimension of the nucleus and on the nature of the phenolic precursors. 
Their affinity for aqueous solvents thus depends on the number and the 
length of the peripheral hydrophilic chains carrying the free -COOH 
groups of polypeptides, however, with significant acidity of the matrix 
(Andreux 1994). 

The properties of the three humic fractions differ especially with 
respect to their molecular weight, their ultimate analysis, and the number 
of functional groups. Fulvic acids have the lowest molecular weight, 
contain more oxygen but less carbon and nitrogen than the other two. The 
functional groups containing oxygen (C0 2 H, OH, C=0) have a rate per 
unit of weight that is higher in fulvic acids than in humic acids and humin 
(Stevenson 1982). 

Humic acids are also extractable from charcoal (Lawson and Stewart 
1989) and research using 13 C nuclear magnetic resonance spectroscopy 
(cf. Sect. 12.3.4) has shown that the stable carbon of Australian soils is 
mainly charcoal (Skjemstad et al. 1996). Although structural models of 
humic molecules have been proposed (e.g. Schulten 1995, Schulten and 
Schnitzer 1997, Schulten and Leinweber 2000, Schulten 2002, Lodygin 
and Beznosikov 2003), most of the concepts concerning molecular 
structure are not applicable to humic acids (McCarthy 2001). Further 
research is needed to obtain precise information about these molecules, 
their bonds with inorganic (Schulten and Leinweber 2000) and xenobiotic 
(Piccolo et al. 1999) molecules and their distribution in different soil 
types, which, in turn, will improve understanding of their role in the 
environment. All modern instrumental methods are needed for these 
researches (Hatcher et al. 2001, Piccolo and Conte 2003). 



12.2 Classical Techniques 



12.2.1 Fractionation of Humic Compounds 



Principle 

For many years humic acids were characterized according to the degree 
they bonded with clays (Springer 1938, Tiurin 1951, Duchaufour 1954, 
1956). 



402 Organic Analysis 

Brown or free humic acids were considered to be only slightly bonded 
to clays, to have the lowest molecular weight and to be relatively 
insensitive to the flocculating action of electrolytes; they were said to 
derive from the oxidation of lignin under the action of the 
polyphenoloxidase and to characterize acid soils in particular, e.g. forest 
soils in a wet climate (Duchaufour 1956). 

Grey humic acids are darker in colour, form closer bonds with mineral 
colloids, have more condensed molecules, flocculate easily in the 
presence of electrolytes; this type of humus is characteristic of black 
soils, but is also relatively abundant in all soils that are rich in calcium. 

Early methods of fractionation of humic acids were thus based on the 
properties of the two main types of compounds: extraction in the presence 
of a flocculating agent in the case of brown humic acids followed by 
washing the residue in water in the case of grey humic acids (Duchaufour 
1956), direct soda extraction of brown humic acids then extraction of two 
fractions of grey humic acids as a function of their bonds with calcium or 
iron and aluminium (Duchaufour 1957). 

To simplify the procedure, Duchaufour and Jacquin (1966) proposed a 
method with electrophoretic fractionation of humic acids on 
pyrophosphate extracts (cf. "Method of Dabin (1976)" Chap. 11). This 
method was compared with that of Tiurin (1951) by Dabin and Thomann 
(1970); it was widely used in France, in particular in IRD 1 laboratories 
(Ratsimbazafy 1973, Dabin 1980). This is the first fractionation method 
described later. The technique of fractionation of humic acids by 
exclusion chromatography (Bailly and Margulis 1968, Bailly and Tittonel 
1972) is also described later, along with a fractionation procedure for 
fulvic acids. Other complex techniques for fractionation of humic 
compounds are described more briefly in Sect. 12.3.1. 

Equipment 

- Plastic electrophoresis tank with three compartments (Fig. 12.2) 

- stabilized supply of direct current adjustable between and 600 V 

- photoelectric densitometer to read the electrophoresis diagrams 

- columns for liquid chromatography, diameter: 2.5 cm, length: 80 cm 
-UV-visible detector equipped with a recorder or a system for data 

acquisition 

- fraction collector (optional) 

- peristaltic pump with flow of 50 mL h" 1 (optional). 



IRD = Institute of Research for Development (ex-Orstom), Bondy, France. 



Characterization of Humic Compounds 



403 



Products 

- Filter paper tapes (Arch 302 or Whatman no. 1 or 2), 5 cm in width and 
35 cm in length, cut perpendicular to the direction the filter is filled 
(when a square sheet of paper is held by one side, the direction 
perpendicular to filling is where the paper curves most under its own 
weight) 

- extraction solutions (see Sect. 1 1.2.1 in Chap. 1 1) 

- buffer solution for electrophoresis: in a 2 L volumetric flask add 13.6 g 
of monobasic potassium phosphate, 2.5 g of soda pellets and 
approximately 1.5 L of inorganic water (cf. Sect. 11.2.1 of Chap. 11), 
adjust pH to 7.4 with soda if necessary, agitate well and complete to 
2L 

- standard dextrane gel Sephadex G25 for molecular weights <5,000 

- standard dextrane gel Sephadex G75 for molecular weights <50,000 

- standard dextrane gel Sephadex G200 for molecular weights <200,000 

- polyvinylpyrrolidone 




Paper tape 




Cathode 



35 cm 



Fig. 12.2. duan tank for paper electrophoresis of humic acids 



-TRIS buffer (pH 9, ionic force 0.5): mix 414 mL of M 2-amino-2 
hydroxymethyl-propane-1, 3-diol, 50 mL of 1 mol (HC1) L _1 solution 
and complete to 1 L inorganic water 

-Borax buffer (pH = 9.1, ionic strength = 0.075): 0.025 mol (Na 2 B 4 7 ) 



404 Organic Analysis 

Procedure for Electrophoresis Fractionation ofHumic Acids 

- Take a quantity of humic extract solution (pyrophosphate or soda) 
corresponding to 25 to 50 mg carbon (cf. titration in Sect. 11.2.2 of 
Chap. 11). 

- Precipitate the humic acids at pH 1 with sulphuric acid, centrifuge and 
wash the centrifugation pellet several times with a 1 mol (V2H2SO4) L" 1 
solution. 

- Dissolve the humic acids in approximately 1 mL of normal soda 
solution so as to obtain a rather thick solution but without solids (cf. 
purification in Sect. 11.2.4 of Chap. 11) and with a homogenous 
concentration of carbon; store in a well-stopped hemolysis tube. 

- Fill the two external compartments of the electrolysis tank to the same 
level with electrophoresis buffer solution; wet the paper strip in the 
buffer solution, dry it between two sheets of filter paper and place it in 
the electrophoresis tank, pull it tight between the two external 
compartments as shown in Fig. 12.2. 

-With a 100 mm 3 micropipette deposit approximately 40 mm 3 of humic 
solution at a distance of 5 cm from the cathode following a straight line 
down the centre of the strip of paper and leaving 1 cm free on each side 
of the paper; in the case of very concentrated solutions, the deposit can 
be reduced to 20 mm 3 . 

- Place the lid on the tank and start the electrical current; regulate the 
current at 10 V per cm of paper, or approximately 200 V; the intensity 
of the current depends on the number of paper strips for simultaneous 
electrophoresis and on the conductivity of the electrolyte (approximately 
15 mA with four strips). The negatively charged humic molecules 
migrate towards the anode; the smaller the molecules, the faster they 
migrate (e.g. brown humic acids, BHA). Migration is rapid at the 
beginning then slows down. The time needed for standard 
electrophoresis is 3 h which corresponds to 10-12 cm displacement by 
brown humic acids. 

- Shut off the current and rapidly remove the paper strips, place them on 
a flat surface and dry them under IR radiation or in the drying oven at 
60°C. Record the radiation transmission of each strip of paper with a 
photoelectric densitometer. 

- An electrophoresis diagram (Fig. 12.3) is obtained in which: 

(1) grey humic acids (GHA) often display a distinct narrow peak up to 1 
cm from the starting line; in the case of the chernozems, Duchaufour 
and Jacquin (1963) reported a second peak which could migrate up to 2 
cm from the starting line; in tropical soils, GHA are frequently spread 



Characterization of Humic Compounds 



405 



out over 3 or even 4 cm with either a single peak, or two or more peaks; 
it is sometimes rather difficult to detect the limit of GHA and their peak 
limit has consequently been arbitrarily fixed at 1/3 of the overall length 
of the diagram, i.e. 3-4 cm for a length of 9-12 cm 



GHA IHA 




Fig. 12.3. Examples of electrophoresis diagrams of humic acids extracted with a 
pH 9.8 pyrophosphate solution in a range of tropical soils (Dabin 1980). 
On the left: direct readings with the optical densitometer at wavelengths 
of 512 nm (top curve) and 625 nm. On the right: corresponding integral 
curves giving in the y-coordinate surfaces corresponding to grey humic 
acids (GHA: from to 1/3 of the graph), intermediate humic acids (IHA: 
between 1/3 and 1/2), brown humic acids (BHA: more than Y 2 of the 
graph), a: tropical podzol (histic tropaquod), A horizon, b: tropical podzol 
(histic tropaquod), B horizon, c: humiferous ferrallitic soil (umbriorthox), 
surface horizon, d: humiferous ferrallitic soil (umbriorthox), subsurface 
horizon, e: weathered tropical soil (oxictropudalf). 



(2) the humic compounds located between 1/3 and 1/2 of the length of 
the diagram are called intermediary humic acids {IHA) 



406 Organic Analysis 

(3) the brown humic acids (BHA) are spread out between the middle 
and the other end of the diagram. 

- The surface area of each fraction is determined; SG is the surface of the 
GHA, 57 that of the IHA, SB that of the BHA, ST is the total surface area 
and % qt is the total quantity of humic acids; the total quantity of each 
fraction is expressed by: 

GHA % = qt SG/ST 
IHA % = qt SI/ST 
BHA % = qt SB/ST 
Figure 12.3 shows some examples of electrophoresis diagrams 
obtained with this method. 

Remarks 

Any liquid circulating on the paper strips can also cause migration of 
humic acids. This should be avoided by making sure that the liquid is at 
the same level at the anode and cathode (otherwise there is a risk of 
siphoning by the paper), and by avoiding evaporation on the paper. 
Evaporation can cause the liquid to move by aspiration on both sides of 
the paper strip. Thus, even without electrical current, certain humic acids 
may migrate and be found in the centre of the strip. With electrical 
current, migration will stop when the speed of migration due to the 
electric potential is equal to the speed of the liquid circulating in the 
opposite direction. It is thus imperative to use a lid to limit evaporation; 
the lid should be shaped like a roof to prevent drops of condensation 
falling onto the paper strips. 

The passage of the current enriches the cathodic compartment in the 
soda and raises its pH; in the case of several electrophoresis series this 
phenomenon can be compensated for by reversing the direction of the 
current and thus of the deposit. 

Procedure for Humic Acid Fractionation on Dextrane Gels 

Fractionations can be carried out in a simple way with elution using 
inorganic distilled water (Bailly and Margulis 1968, Bailly and Tittonel 
1972): 

- prepare a concentrated solution of humic acids in the same way as for 
electrophoresis but with a sample specimen corresponding to 2-10 mg 
carbon, diluted in 3-10 mL of 1 mol (NaOH) L" 1 solution 

- fill the columns with the Sephadex gel: 50 cm for G25 and G75, 70 cm 
approximately for G200, these heights are likely to vary with the type 
of humus (Bailly and Tittonel 1972) 



Characterization of Humic Compounds 



407 



deposit the test specimen on the G25 column and elute with inorganic 

water either by gravity (descending chromatography), or with a 

peristaltic pump at a flow of approximately 30 mL h" 1 (ascending 

chromatography) 

carry the effluent in a UV photometer detector regulated at 253.7 nm; 

record the chromatogram and collect the fractions corresponding to the 

main peaks (Fig. 12.4); 



T% 



100 




130Fractions 10 

T% 








V 


IV 




25 




( 




III 


50 








\ I 


75 








\ I' 


100 


— 









NaOH 



50 



VII! 



90 



130 Fractions 



25 

5Q 
75 

100 





10 




50 90 




25 


k 


50 






j 


75 
100 


-^ 




IX ' 



10 



50 



90 Fractions 




130 Fractions 



130 Fractions 



Fig. 12.4. Examples of separation by exclusion chromatography on dextrane gel 
(Bailly and Tittonel 1972). On the left, grassland podzolic soil, A1 
horizon, 0-6 cm depth; on the right, forest grey soil, A2 horizon, 
depth 16-29 cm. a: fractionation of the humic acid extract on Sephadex 
G25 F, test specimen corresponding to 4.7 mg C, b: fractionation on 
Sephadex G75 of peak V obtained in a, c: fractionation on Sephadex 
G200 of peak VIII obtained in b. 



the first eluted peak corresponds to the largest molecules that were not 
separated by gel exclusion; collect this fraction and subject it to 
exclusion chromatography in a similar way but on column G75; 
fractionate the new first peak eluted on this column again on column 



408 Organic Analysis 

G200; Fig. 12.8 presents two series of examples of chromatograms 

obtained successively on the three types of columns with two soils. 

This type of fractionation by gel permeation can also be performed in 

buffered solutions to avoid interactions between the gel and the solution, 

e.g. Tris and Borax buffers prepared in the same way as in "Products" in 

Sect. 12.2.1 (Cameron et al. 1972a). 

Fractionation of Fulvic Acids 

The method of Lowe (1975) is a very simple way to separate fulvic acids 
into two fractions: a coloured polyphenols fraction and an almost 
colourless fraction with a prevalence of polysaccharide. Since C h , C f , C a 
are carbons in humic acids, fulvic acids and their polyphenols coloured 
fraction respectively, the C^:Cf and C a :Cf ratios were linked to the types 
of horizons used in the Canadian soil classification system which 
facilitate the distinction between some of these types (Lowe 1980): 

- treat a fraction of the fulvic acid extract using 1 g polyvinylpyrrolidone 
for 100 mL of solution 

- agitate intermittently for 30 min and filter 

-titrate carbon on the filtrate (cf. Sect. 11.2.2 of Chap. 11) to quantify 
the Ca fraction; the fulvic acid sample must be sufficient in volume to 
allow titration with acceptable precision. 

12.2.2 Titration of the Main Functional Groups 



Principle 

The procedures described here are based on the measurement of total 
acidity and of carboxylic acidity of Wright and Schnitzer (1959) and of 
Schnitzer and Gupta (1965). 

For measurement of total acidity, the sample is treated with a barium 
hydroxide solution under N 2 for 24 h. The Ba(OH) 2 remaining in the 
solution after the reaction is then back titrated with a standard acid 
solution. 

For the titration of carboxylic groups, the humic materials are agitated 
for 24 h with calcium acetate solution in excess which causes the release 
of acetic acid according to a reaction of the type: 

2 RCOOH + (CH 3 COO) 2 Ca -> (RCOO) 2 Ca + 2 CH3COOH 



Characterization of Humic Compounds 409 

The acetic acid released is then titrated with a standard soda solution. 

The proportion of phenolic groups is calculated by the difference 
between total acidity and the acidity of the carboxylic groups. 

Other measurement techniques for functional groups (phenolic, 
alcoholic, ketonic, quinoid) are described briefly in Sect. 12.3. 

Equipment 

- 125 mL Erlenmeyer flasks with screw caps 

- titrimeter equipped with a combined electrode for the measurement of 
pH. 

Products 

- 0.2 mol (1/2 Ba(OH) 2 ) L" 1 barium hydroxide solution: weigh 31.548 g 
of Ba(OH) 2 ,8H 2 (quality containing the minimum of carbonate), 
dissolve in inorganic C0 2 -free water, complete to 1 L and protect from 
atmospheric C0 2 with a trap containing soda lime 

- 0.5 mol (HC1) L 1 hydrochloric acid solution: prepare using standard 
commercial dose; dilute with inorganic C0 2 -free water. 

- 1 mol (l/2Ca(CH 3 COO) 2 ) L" 1 calcium acetate solution: dry the pure 
product at 105°C and weigh 79.085 g under anhydrous atmosphere, 
dissolve in inorganic water and complete to 1 L. 

-0.1 mol (NaOH) L" 1 soda solution: prepare using standard commercial 
dose; dilute with inorganic water, complete to 1 L and protect with a 
trap containing soda lime during storage. 

Procedure 



Total Acidity 

To obtain the maximum number of exchangeable sites in acid-active 
form, it is advisable to work with carefully purified humic materials in 
order to reduce their ash rate (cf. Sect. 1 1.2.4 of Chap. 1 1). 

Place an exact weight of between 50 and 100 mg of freeze-dried humic 
material in a 125 mL Erlenmeyer flask with a screw cap and add exactly 
20 mL of 0.2 mol (Ba(OH) 2 ) L" 1 solution. Perform a blank assay 
containing only the 0.2 mol (Ba(OH) 2 L" 1 solution without the sample. 
Put the flasks under nitrogen atmosphere, stop well and agitate for 24 h at 
room temperature. Filter the suspension, wash the residue well with 
distilled CC^-free water, add the washing waters to the filtrate and carry 
out potentiometric titration of the resulting extraction solution with 0.5 



410 Organic Analysis 

mol (HC1) L" 1 solution up to pH 8.4. V h and V are the volumes of 
standard acid solution for titration of the blank assay and of the sample, 
respectively, N & is the acid normality and P the weight of the sample 
(mg); total acidity A t in milliequivalents per gram of humic material is 
expressed by: 

A t =l,000(V b -V s )N a /P 

Carboxyl Groups 

Place in a 125 mL Erlenmeyer flask with a screw cap an exact weight of 
between 50 and 100 mg of humic material; add 10 mL of 1 mol Vi 
Ca(CH 3 COO) 2 L" 1 solution and 40 mL of inorganic CC^-free water. At 
the same time, perform a blank assay containing the reagents without the 
humic sample. After 24 h of shaking at room temperature, filter the sus- 
pension, rinse the residue with distilled CC^-free water, combine the 
filtrate and washing water and perform potentiometric titration with the 
0.1 mol (NaOH) L" 1 solution up to pH 9.8. If V s and V h are the volume of 
titrating solution for the sample and blank assay, respectively (mL), N b is 
the normality of the standard soda solution (mol L" 1 ), P the sample 
weight (mg); the carboxylic acidity A c in mol (COOH) g" 1 humic 
material, is expressed by 

A c = l,000(V s -V b )N h /P. 

Phenolic Acidity 

This A v acidity can be expressed in mol (phenolicOH) g" 1 humic material 
by 

Ap — A\—A c . 

12.2.3 UV-Visible Spectrometry 



Principle 

Among other molecular spectrometry techniques, UV spectrometry 
records electronic energy transitions in the molecules whereas lower- 
energy infra-red radiation records variations in molecular kinetic energy. 

Although absorption in the ultraviolet and visible field of the 
electromagnetic spectrum does not give a band that is very characteristic 
of humic compounds (Schnitzer and Kahn 1972, Schnitzer 1978), the 
E4:E6 ratio of absorbance at 465 (E4) and 665 nm (E6) is often used to 
characterize humus. Ratios lower than 5 are characteristic of humic acids 



Characterization of Humic Compounds 



411 



while fulvic acids have higher ratios; this ratio is independent of the 
concentration of humic materials but is not the same for humic materials 
extracted from different types of soils. 



CD 

UJ 

a 




UJ 

LU 





1 




— 1 — 




r 


6 




b 










5 






~~~*~""-^ " 


4 


" 


* - 


^*^^^ 


3. 


, l. 




. . 



CO 
LU 



6 8 10 12 
PH 



LU 
8 

7 


-1 1 

I 9 

' f 3 


i 1 1 — 

To 


6 


*2 


. 


5 


— i j i 


j- * 



14 1.8 2.2 

Free radicals x 10 l7 g' 1 



10 pH 



Fig. 12.5. Effect of certain factors on the value of the E4:E6 ratio (Chen et al., 
1977): (a) effect of the pH in different fractions of a fulvic acid (Bh 
horizon of a Canadian podzol) separated on Sephadex gel; the 
molecular weights measured by osmometry (Hansen and Schnitzer, 
1969) were: 883 for AF-IV, 1181 for AF-III, 1815 for AF-II, 2110 for AF-I 
and >21 10 for >AF-I, (b) effect of pH in some humic acids of Ah 
horizons of Canadian soils from the area round Alberta: Black 
Chernozem, Solod, Solonetz, (c) effect of the concentration of free 
radicals measured by electron spin resonance (cf. Sect. 12.3.6) for the 
fulvic acid used in a, (the numbers on the curve are pH values). 



Kononova (1966) believed that the E4:E6 ratio was related to the 
degree of condensation of the aromatic carbon lattice, a weak ratio 
indicating a high degree of condensation of the aromatic humic 



412 Organic Analysis 

components, a strong ratio indicating the presence of a higher proportion 
of aliphatic structures. 

Chen et al. (1977) made a thorough study of the information provided 
by the E4:E6 ratio. Their study showed that the E4:E6 ratio: 

- is mainly governed by molecular size (or molecular or particle weight; 
Fig. 12.5a, b) 

- is highly affected by the pH (Fig. 12.5a, b) 

-is correlated with the concentration of free radicals (Fig. 12.5c), and 
with O, C, CO2H contents and total acidity (these measurements are 
also correlated with the size of the particles) 

- does not present a direct correlation with the concentration of 
condensed aromatic nuclei, which would invalidate the assumption of 
Kononova (1966) 

- is independent of the concentration of humic or fulvic acid, at least in 
the field of 100-500 ppm, which confirms the other assumption of 
Kononova. 

In agreement with Kononova (1966), these authors finally showed that 
the most favorable range of pH to measure E4:E6 ratios is between 7 and 
8. This can be obtained by dissolving humic material in a 0.1 
mol (NaHC0 3 \ L 1 solution at a concentration of between 200 and 400 
mgkg" 1 . 

Ghosh and Schnitzer (1979) proposed a mechanism linking the 
macromolecular characteristics of humic substances and UV-visible 
absorption: optical density decrease with an increase in the concentration 
of neutral electrolyte, indicating a reduction in the size of the particles 
probably due to a rolling up of the macromolecule. 

Equipment 

-UV-visible spectrograph with an adjustable double beam and fixed 
wavelength (465 and 665 nm) or preferably with variable wavelength 
between 200 and 700 nm 

- quartz tanks for UV spectrometry. 

Reagents 

- 0.05 mol (NaHC03) L" 1 solution: in a 1 L volumetric flask dissolve 
4.200 g of NaHC03 (quality suitable for spectrography) in inorganic 
distilled water, complete to 1 L and stop the flask well before storage. 



Characterization of Humic Compounds 413 

Procedure 

Dissolve 2-4mg of humic material in 10 mL of the 0.05 
mol (NaHC03) L" 1 solution. Check the pH after dissolution, it should be 
close to 8 (the pH of a 0.05 mol (NaHCC^) L" 1 aqueous solution is 8.3). 
Fill the quartz measurement tank to mid-height with this solution and fill 
the reference tank with a pure 0.05 mol (NaHCC^) L" 1 solution. Measure 
the absorbance at 465 and 665 nm. The ratio of these two absorbencies is 
the E4:E6 ratio. 

If more detailed studies are required, the optical density (OD) spectrum 
can be recorded in the 200-350 nm UV range (Ghosh and Schnitzer 
1979) or in the 400-700 nm range; in the latter case, the straight lines 
Log(OD) =/(Log k) can be plotted and, according to Chen et al. (1977), 
its slope should be equal to -6.435 Log(E4:E6) 



12.2.4 Infra-Red Spectrograph^ 



Principle 

The infra-red spectrum between 1 and 100 |Lim wavelength makes it 
possible to observe the vibrations of stretching and deformation of the 
molecules (as the spectrum of molecular rotation corresponds to less 
energetic radiations of wavelengths higher than 100 |Lim). In practice, the 
most useful spectral field for organic chemistry is in medium IR between 
the two wavelengths A of 2.5 and 15 |Lim corresponding to wavenumbers 
1/X between 4,000 and 660 cm" 1 (cf. Sect. 5.1.1 of Chap. 5). The near IR 
zone can also be widely explored with the help of chemometrical 
software (cf. Sect. 5.3.1 of Chap. 5). 

In humic substances, the IR spectrum mainly reflects oxygenated 
functional groups such as -C0 2 H, -OH and C-O. Some IR bands are 
particularly well defined (Schnitzer 1971) at wavenumbers 3,400 cm" 1 (H 
bound to OH), 2,900 cm" 1 (aliphatic CH bonds), 1,725 cm" 1 (C-0 of 
C0 2 H, C-0 elongation of ketonic carbonyls), 1,630 cm" 1 (aromatic C-C, 
H linked to C-0 of carbonyls, COO"), 1,450 cm" 1 (aliphatic CH), 1,400 
cm" 1 (COO", aliphatic CH), 1,200 cm" 1 (CO stretching, OH deformations 
of C0 2 H), 1,050 cm" 1 (Si-0 of the silicated impurities). 

The IR spectrum does not provide much information on the chemical 
structure of the core of humic substances. However, it is very useful for 
preliminary characterization of humic materials of different origin (Fig. 
12.6) to determine the effect of different extractions or chemical 



414 



Organic Analysis 



purification agents (cf. Sect. 11.2.4 of Chap. 11), and to study the 
reaction of derivatisation such as silylation, methylation, and acetylation. 
The IR spectrum also makes it possible to detect changes in the 
structure of humic materials following oxidation, pyrolysis or other 
treatments. Lastly, it is a practical method to characterize the formation of 
metal-humate and clay-humate complexes or to detect interactions 
between humic materials and other organic molecules such as pesticides. 







E 

V) 

c 
TO 




4000 



2000 



1000 500 4000 



2000 



1000 500 



Wavenumber (cnV 1 ) 



Wavenumber (cm 1 ) 



Fig. 12.6. hfra-red spectra of some fulvic (on the left) and humic (on the right) 
acids of standard and reference samples of IHSS (Senesi et al. 1989): 
(a) Suwannee River Standard, code IHSS 1S101F(AF) and 1S101H 
(AH),(a1) Suwannee River Reference, code IHSS 1R101F (AF) and 
1R101H(AH), (b) Nordic aquatic, code 1R105F and 1R105H, (c) Soil 
St, code 1S102Rand 1R102H, (d) Peat, code 1R103Fand 1R103H, 
(e) Leonardite, code 1R104H. 

Great care should be taken in interpreting the spectra and particularly 
to avoid confusing the organic or mineral origin of the absorption bands 
(Russel and Anderson 1977). 

Equipment 



Double beam IR absorption spectrograph with a field frequency 
between 300 and 4,000 cm" 1 



Characterization of Humic Compounds 415 

-manual hydraulic press for the preparation of pellets for IR 
spectrography (e.g. standard 12 ton Spex-Carver) 

- polished stainless steel vacuum pelletizer, diameter 13 mm. 

NB: it is also possible to work without a pelletizer or a hydraulic press; 
spectra can be obtained in suspensions that are maintained between two 
IR-transparent blades; spectra can also be obtained in solutions with a 
solvent that is transparent to IR. The technique described below is not the 
cheapest but has been shown to be particularly suitable for IR 
spectrometry. 

Product 

- Potassium bromide in powder form for IR spectrometry. 

Procedure 

With the agate mortar, prepare a KBr pellet by mixing 1 mg of humic 
material with 400 mg of dry KBr. Place the powder in the pelletizer, put 
it under vacuum and press with a pressure of 7,600 kg cm" 2 for 20 min 
(cf. "Preparation of Discs (Solid Solution)" in Chap. 5). Unmould the 
pellet, which should be vitrified, and place it in the measuring cell of the 
IR spectrometer, put a pellet of pure KBr in the reference cell and record 
the spectrum between 300 and 4,000 cm" 1 . 

12.3 Complementary Techniques 



12.3.1 Improvements in Fractionation Technologies 

The techniques described later are the result of improvements in 
electrophoresis and gel exclusion chromatography (cf. Sect. 12.2.1). 

Electrophoresis - Electrofocusing Method 

Cacco et al. (1974) proposed an improvement of the electrophoresis of 
humic compounds by using the electrofocusing method described by 
Righetti and Drysdale (1971). In this technique, humic compounds 
migrate from anode to cathode in a polyacrilamide gel in the presence of 
ampholines which cause a pH gradient. The migration stops when each 
compound reaches its isoelectric point. Figure 12.7 shows the results of 



416 



Organic Analysis 



isoelectrophoretic characterisation (Cacco and Maggioni 1976) of fulvic 
and humic acids extracted with pyrophosphate at pH 7 from an alpine 
podzol. Rusina et al. (1983) suggested a system of calculation of 
molecular parameters by electrophoretic mobility in the polyacrylamide 
gel. Electrophoresis techniques are generally used for the study of 
molecular size but also of the electrical charge of humic substances 
(Duxbury 1989). 




cm 6 4 2 



cm 6 4 2 o 



Fig. 12.7. Isoelectrophoretic characterization of fulvic (deep Bh horizon) and humic 
(surface A1 and deep Bh horizons) acids extracted from an alpine podzol 
(Cacco and Maggioni 1976) 



Gel Exclusion Chromatography 



The gel can be calibrated with the molecular weights of humic acids 
measured jointly using other techniques. Figure 12.8 shows the 
calibration curves obtained by Cameron et al. (1972a) with four types of 
gel; the molecular weights (on the x-coordinate) were measured by a 
sedimentation technique using ultracentrifugation (Cameron et al. 1972b). 
In such studies, the gel is characterized by the K ay parameter suggested by 
Laurent and Killander (1964): 

K aY = (V R -V )/(V-V ), 

Where is the V R : volume of retention, V : volume of pores and V t : 
volume of total column. 



Characterization of Humic Compounds 



417 



The median values of K ay are adjusted to the median molecular weights 
M(Fig. 12.8) by means of two constants k\ and ^2 according to law: 
K aY = ki \nM+k 2 . 




Kav 

0.8 

0.6 

0.4 

0,2 



Kav 

1 






10 1 M 10* 10 1 




10* M 10' 



Fig. 12.8. Calibration of four types of gel (Cameron et al. 1972a) for the 
fractionation of humic acids in comparison with other macromolecular 
compounds (solid lines and solid circles: humic acids, dotted lines 
and open circles: data from the protein fractionation, sucrose is used 
to determine the stationary liquid volume V t See text for K aY and M: 
(a) Sephadex G100 in tris buffer (cf. Sect. 12.2.1 for preparation), (b) 
Sephadex G100 in borax buffer (cf. Sect. 12.2.1 for preparation), (c) 
Biogel P-150 in tris buffer and (d) Sepharose 6B in tris buffer. 



Nowadays, fractionation by gel permeation can be carried out by high 
pressure liquid chromatograph (HPLC) and in addition, new gels are 
more powerful than the older Sephadex gels. For example the "Zorbax 
PSM 1000" silica gel used by Morizur et al. (1984) allowed better 
recovery of all the organic matter with approximately 1/100 the ion 
exchange capacity of Sephadex gel. 

Beckett et al. (1987) used a flow field-flow fractionation (flow FFF) 
technique for fulvic and humic acid fractionation, which appears to be a 
powerful tool to obtain information on molecular weights. 



418 Organic Analysis 

12.3.2 Titration of Functional Groups 

Section 12.2.2 described titration techniques for the main functional 
groups of humic compounds, carboxylic and phenolic groups, the latter 
being obtained by calculating the difference between total acidity and 
carboxylic acidity. 

Chemical titration can be used for other functional groups. The main 
groups that have been the subject of such investigations are: 

- Total OH groups can be determined by acetylation of humic 
substances with acetic anhydride in pyridine (Schnitzer and Skinner, 
1965): 

2 ROH + (CH 3 CO) 2 -► CH3COOR + CH3COOH 

The acetylated humic substances are then carefully isolated from the 
reactional medium. Hydrolysis in alkaline medium releases acetates from 
the acetyl groups; distillation in strong acid medium enables recovery of 
acetic acid which is titrated by acidimetry. The number of moles of acetic 
acid collected corresponds to the number of total OH groups of the humic 
substance. 

-Alcoholic OH groups can be estimated by "total OH groups" minus 
"phenolic OH groups". However, as the phenolic OH groups are 
themselves calculated by difference (cf. Sect. 12.2.2), it is advisable to 
take the laws of propagation of errors into account. 

- Total C-O Groups can be quantified by the reaction of humic 
substances with hydroxylamine chlorhydrate in 2-dimethylaminoethanol 
medium: 

R^C-O + NH 2 OHHCl -> R^C-NOH + H 2 + HC1 
Excess hydroxylamine hydrochlorate is back titrated with a standard 
HCIO4 solution (Fritz et al. 1959). 

- Quinonic C-O groups can be titrated by reduction in OHphenolic 
groups by ferrous iron in triethanolamine; the excess ferrous iron is back 
titrated by amperometry with a dichromate solution (Glebko et al. 1970). 

- Ketonic C-O groups can be estimated by the difference between total 
C-O groups and quinonic C-O groups. 

In addition, Schnitzer (1978) cited several attempts to characterize acid 
groups by direct potentiometric titration. It was difficult to clearly 
distinguish the two main types of acidity (OH and C0 2 H functional 
groups) using this method even in a non-aqueous medium. Certain 
authors did succeed including Rosell et al. (1972), who simultaneously 
measured three types of acidity by potentiometric titration in 80% 
TV-methyl acetamid aqueous solution. 



Characterization of Humic Compounds 419 

De Nobili et al. (1990) presented an alternative to the technique 
described in Sect. 12.2.2 for the determination of carboxylic groups: the 
precipitation of humic substances with cethyltrimethylammonium cation 
detergent. 

12.3.3 Characterization by Fragmentation 

One of the ways to study heavy humic macromolecules consists in 
splitting them up and identifying the fragments. These methods can be 
classified in four main groups: oxidative fragmentation, reducing frag- 
mentation, other chemical degradation techniques and thermal fragmenta - 
tion by pyro lysis. 

Oxidative Fragmentation 

This group of techniques can be divided into two subgroups, (i) oxidation 
with permanganate and (ii) other oxidative techniques. 

Oxidation with permanganate was widely used on humic materials 
from different types of soils: e.g. Ah horizons of Solonetz, Solod and 
Chernozem (Kahn and Schnitzer, 1971a; Kahn and Schnitzer, 1972b), 
forest grey soil under different farming systems (Kahn and Schnitzer, 
1972b), tropical volcanic soils (Griffith and Schnitzer, 1975), mediterranean 
soils (Chen et al. 1978a), non-hydrolysable humic residues (Ogner, 1973) 
and fulvic acid fractions (Khan and Schnitzer, 1971b). 

The analytical procedure varies with the author. The humic materials 
can first be fractionated or subjected to a derivatisation reaction 
(methylation) before oxidation. Kahn and Schnitzer (1971b) oxidated 1 g 
of humic material by boiling at reflux for 8h with 250 mL of 4% KMn04 
aqueous solution. The excess of permanganate must be destroyed by 
controlled addition of small volumes of methanol and the solution 
removed from insoluble Mn0 2 by filtration and rinsing. The acidified 
filtrate is then extracted with ethyl acetate in a liquid-liquid extractor for 
48h. The extract is brought to dry in the rotary evaporator, dissolved in a 
small volume of methanol and methylated with a diazomethane solution 
in ether. The end products are then split by chromatography and 
identified with the usual range of spectrographic methods used for 
molecular characterization (UV, IR, mass and NMR spectrometry), the 
most widely used method being gas chromatography coupled with mass 
spectrometry (GC-MS). 



420 Organic Analysis 

Other oxidative reagents have been used for studies of the degradation 
of organic matter. Neyroud and Schnitzer (1974) then Griffith and 
Schnitzer (1976) studied the products of alkaline oxidation of humic and 
fulvic acids by cupric oxide. Oxidation is performed in an autoclave at 
170°C on lg of humic product mixed with 100 mL of NaOH 2 mol L" 1 
solution and 5g CuO; the end of the procedure is almost the same as for 
permanganate oxidation (see earlier part in this section). 

Other methods included nitrobenzene alkaline oxidation (Morrison 
1963), nitric acid oxidation (Hansen and Schnitzer, 1967), hypohalogenite 
oxidation (Chakrabartty et al. 1974) and peracetic acid oxidation 
(Schnitzer and Skinner, 1974). Griffith and Schnitzer (1989) reviewed 
oxidative degradation techniques, one of the analytical tools for the study 
of humic substances (Hatcher et al. 2001). 

Reductive Fragmentation 

The most commonly used reagent was sodium amalgam (Mendez and 
Stevenson 1966; Stevenson and Mendez 1967; Piper and Posner 1972) 
but other reducers were also tested such as zinc powder (Hansen and 
Schnitzer 1969). Stevenson (1989) reviewed reductive fragmentation 
techniques. 

Other Degradative Chemical Methods 

Boiling humic acids in water releases polysaccharides and small 
quantities of phenolic acids and aldehydes, polypeptides, alkanes and 
fatty acids (Neyroud and Schnitzer 1975). 

Acid hydrolysis at reflux boiling enables between 1/3 and 1/2 of 
organic matter to be dissolved in most soils (Schnitzer, 1978) but this 
technique was mostly widely used for the study of the organic forms of 
nitrogen (cf. Sect. 14.2.1 of Chap. 14); Anderson et al. (1978) studied 
ether-soluble products of acid hydrolysis of fulvic and humic acids. 

Alkaline hydrolysis was also used as a degradation method for the 
study of humic molecules. Neyroud and Schnitzer (1975) subjected 
humic materials to four successive series of hydrolysis with a-NaOH 2N 
solution in an autoclave at 170°C for 3 h. The recovery and identification 
of the hydrolysed products was accomplished using techniques similar to 
those of the other degradation methods (cf "Oxidative Fragmentation"). 
Parsons (1989) analysed the main fragmentation mechanisms in the acid 
and alkaline hydrolysis of organic materials. 

A method developed for depolymerization of coals (Ouchi and Brooks 
1967) was applied to degradation of humic acids (Jackson et al. 1972): 



Characterization of Humic Compounds 



421 



reaction with phenol in the presence of ^-toluenesulfonic acid as catalyst. 
A review of degradation techniques by phenol and sodium sulphide was 
published by Hayes and O'Callaghan (1989). Cheshire et al. (1968) 
studied the effect on humic acids of alkaline fusion after acid boiling. 

Thermal Degradation 

Thermogravimetry (TG), differential thermogravimetry (DTG), differential 
thermal analysis (DTA) and isothermal heating were used to explore the 
mechanism of thermal decomposition of humic materials (Schnitzer 
1978). Schnitzer and Hoffmann (1964) studied the chemical evolution of 
humic and fulvic acids under the action of temperatures up to 540°C; Fig. 
12.9 shows the curves of differential thermogravimetry these authors 
observed on their samples. 



o 



2> 
<x> 

o 

CD 
(Z 




100 300 500 
Temperature °C 



00 300 500 



Fig. 12.9. Curves of differential thermogravimetry (Schnitzer and Hoffman 1964) 
of organic materials of a podzol: a: surface 02 horizon, b: deep Bh 
horizon 



Kodama and Schnitzer (1970) used differential thermal analysis in a 
study of the mechanism of thermal decomposition of fulvic acids. Chen 
etal. (1978b) also used this technique to compare the physicochemical 
characteristics of humic and fulvic acids of Mediterranean soils (Fig. 
12.10). 



422 



Organic Analysis 



o 



i 



o 
£ 

n 
o 

■D 

c 





Temperature- 



Temperature 



Fig. 12.10. Differential Thermal Analysis applied to humic (on the left) and fulvic 
(on the right) acids extracted from Mediterranean soils (Chen et al. 
1978): (a) clayey brown soil, (b) sandy brown soil, (c) clayey silty sandy 
red soil, (d) sandy silty red soil, (e) silty sandy red soil, (f) sandy silty 
brown soil 



Characterization of Humic Compounds 



423 



Fig. 12.11. 

Chromatogram of 
pyrolysis products of 
a fulvic acid extracted 
with soda in a deep 
Bh horizon of a podzol 
(Martin, 1976) 




8 12 

Time (rnln) 



§ 



c 
o 

p 



i! 



Soil before extraction 



Vfe 



. 4 ^jL >li .JU J T m. j. ..I. .,i? 



Humfc acids 



II H & 



m 



ukiL 



JtLL 



ijhlllrlii |-i.t , 



J 



Hurnin residue 



he 



i.iAiAilm.Jm 



l t llll]ll11i, l rTl.|ll l . ,.»l.l.l>| l^. 



I.? Ill ij Jill TJTi 



Soil after extraction 

W IK 



tM 



lllilk # J|j[|^ .,;.! 



ii.i....L 



(C HM ii-J >*0 

M/E 



Fig. 12.12. Mass spectra of pyrolysis obtained by Saiz-Jimenez et al. (1979) on a 
sample of brown soil on granite rock (typical xerochrept); the humin 
residue fraction was the result of alkaline extraction of the soil following 
humic acid extraction and complete destruction of silicates by the HF- 
HCI mixture 



424 Organic Analysis 

Pyrolysis-gas chromatography was developed by Kimber and Searle 
(1970) for the study of soil organic matter. Figure 12.1 1 shows one of the 
chromatograms of pyrolysis obtained by Martin (1976) on fulvic acids of 
a deep Bh horizon of podzol. 

The pyrolysis mass-spectrometry described by Meuzelaar et al. (1973) 
was applied to humic compounds by several authors (e.g. Meuzelaar et al. 
1977 Saiz- Jimenez et al. 1979). The humic sample was dispersed in a 
soda solution or in methanol, covered and placed on a ferromagnetic coil 
and pyrolyzed at 510°C. An example of the mass spectrum of the 
pyrolysis products is shown in Fig. 12.12. Reviews by Bracewell et al. 
(1989) and Schulten (1996) give more data on the thermal degradation 
products of humic materials. 

12.3.4 Nuclear Magnetic Resonance (NMR) 



Principle 

The majority of atomic nuclei turn around their axis and thus have an 
angular moment expressed by the formula [h/2%][I(I+l)] 12 where h is 
the Planck constant and / the spin number. The spin number values can 
be 0, 1/2, 1, 3/2, 2, etc., depending on the nature of the nucleus: for ! H 
and 13 C, / = 1/2, but for 12 C, 7=0. Because of the electric charge, the 
rotation of the nucleus creates a magnetic field. Conversely, if a nucleus 
is placed in a magnetic field H it can orient itself in one of the (2 7 +1) 
directions linked to the direction of the field. Each direction corresponds 
to an energy state and it is possible to induce a resonance between the 
energy states by using electromagnetic radiation of a frequency vsuch as: 

v = yH /2%, (12.1) 

where the gyromagnetic ratio ^is a constant that depends on the type of 
nucleus. 

Table 12.1 Relative detectability (RD) of a nucleus in soil organic matter by 
NMR (Wilson 1981) 



Nucleus RD 



! H a 10 2 

27 Al 10 1 

23 Na 10° 



Characterization of Humic Compounds 425 



o 



! H b 10 



55 Mn lO" 1 

29 Si lO" 1 

13 C lO" 3 

14 N lO" 3 

39 K lO" 3 



17 lO" 3 

25 Mg lO" 3 

67 Zn lO" 3 

3i p 10 -4 

43 Ca lO" 4 

57 Fe lO" 5 

15 N lO" 6 



a : very variable, depends on the water 
contents and the pH 

b : H of soil organic matter only 



The atom of hydrogen (1= 1/2) gives (27+ 1) = 2 possible orientations 
of the nucleus and it is possible to detect its resonance. On the other hand, 
most nuclei are not detectable: for 12 C, (27+ 1) = 1 thus there is only one 
possible orientation of the nucleus in the magnetic field and it is not 
possible to induce a resonance. Only the 13 C isotope of carbon can be 
studied but is much less abundant than the 12 C isotope in the natural state. 

Finally, the most important factors in measuring the detectability of an 
element in soil or in soil extracts are the spin number 7, and the gyro- 
magnetic ratio y but also the abundance of the element (n) and the 
relative abundance of the isotope under study (N). Table 12.1 presents the 
detectability of atomic nuclei in soil as a function of a calculation by 
Wilson (1981) with the formula: y 3 NI(I+l)n. This table does not give the 
detectability of an element itself but its detectability in the soil or in a soil 
organic matter medium. Thus, an element that is sensitive to NMR such 
as 31 P will be detected with difficulty because of its low concentration. 



426 Organic Analysis 

The traditional NMR approach consists in subjecting the sample to a 
radio frequency scan (continuous wave NMR), with a fixed magnetic 
field (or vice versa) and recording resonance when the irradiation 
frequency matches the frequency of nuclear transition given by (12.1). 
The Fourier Transform NMR often enables high quality spectra to be 
obtained more rapidly. In FTNMR, the nucleus is subjected to short and 
intense pulsation of radiation and its behaviour is observed. All the nuclei 
resound simultaneously and the resulting spectrum of the signal as a 
function of time (free induction decay, FID) is not very useful for the 
chemist. Signal processing by Fourier transformation should be used to 
obtain more easily interpretable spectra of the continuous wave NMR 
type. To increase sensitivity, a large number of "FID" has first to be 
collected on the computer and then an average used to calculate the 
Fourier transform. 

The NMR technique would provide little useful information for 
structural chemistry if only the spin transition(s) of the nucleus were 
measured at frequencies corresponding to (12.1). In fact, the electronic 
environment of the nucleus protects it from the applied magnetic field 
(H ) and the real magnetic field at constant frequency (or the frequency 
at constant field) necessary for nuclear resonance depends on how 
effectively the nucleus is protected. In organic molecules, the functional 
groups have different electronic distributions and the frequencies of 
resonance of each of their nuclei shift slightly depending on the nature of 
the functional group. It is then possible to identify these groups. In 
practice, the shift in frequency is identified by comparing it to a standard, 
usually tetramethylsilane (TMS), in terms of chemical shift (S) calculated 
by the ratio of the "chemical shift of frequency" compared to the 
"frequency of the standard". As the chemical shifts are weak, 8 are 
generally noted in parts per million: 8(ppm). 

Another significant parameter is relaxation. After the excitation of a 
nucleus, some time passes before it returns in its fundamental energy 
state. In theory, this return occurs in two ways: by interaction with the 
molecular lattice or by spin energy exchange with "brother" nuclei. The 
time constants corresponding to these two processes are named spin- 
lattice relaxation time and spin-spin relaxation time (Tl and T2, 
respectively). This phenomenon is significant for the soil chemist. In 13 C 
NMR studies, it can affect the quantitative measurement of the carbon of 
the functional groups. 



Characterization of Humic Compounds 427 

Following the development of this technique NMR was used for the 
study of soil organic matter. Originally extracts were analysed in solution 
by studying ! H proton NMR then 13 C NMR, first qualitatively, and then 
while trying to quantify the observations. The material had to be soluble 
in a solvent which did not give resonance to the element under study (e.g. 
for studies of ! H, D 2 was used instead of H 2 0), but this is no longer 
indispensable (Wilson, 1981). More recent studies use NMR on the solid 
phase. Wilson (1981, 1987) reviewed these methods and their use for the 
study of soil organic matter. Steelink et al. (1989) provided comple- 
mentary information in the field of ! H and 13 C NMR of humic substances 
in solution. Wilson (1989) and Tate (1998) provided additional theo- 
retical information in the field of solid state NMR and its use for humic 
substances. Simpson (2004) applied coupling NMR and separation 
techniques. 15 N NMR was applied to the study of the nitrogen cycle 
(Thorn and Mikita 2000). 

Study of Humic Materials by 1 H NMR 

Schnitzer and Barton (1963) were the first to observe NMR spectra of 
organic extracts of soil. They used spectrometry with continuous wave 
scanning applied to fractions of methylated fulvic acids which provided 
relatively poor structural information. Subsequently, a relatively large 
number of authors used the same wave scanning technique ! H NMR. 
Lentz et al. (1977) obtained spectra of better quality with the Fourier 
transform technique. Wilson et al. (1978) used the techniques with 
Fourier transform in a high magnetic field at a very high frequency 
thereby further increasing the information provided by ! H NMR spectra 
(Fig. 12.13). 

Study of Humic Materials in Solution by 13 C NMR 

13 C NMR has several advantages over ! H NMR for the structural analysis 
of molecules: it provides direct information on the structural skeleton, 
which enables observation of functional groups without protons like 
ketones. The carbon nucleus provides more significant chemical shifts 
enabling detection of finer differences in molecular structures. The 
signals can also include narrower peaks, and this reduces the risk of one 
peak concealing another. On the other hand, the disadvantage of 13 C 
NMR lies in the small proportion of 13 C isotopes (only 1.1% of carbon) 
leading to difficulty in detecting the signals (Wilson 1981). 



428 



Organic Analysis 



For satisfactory detection, the conditions required to obtain the 13 C 
spectra always have to be optimized. These spectra are almost always 
obtained under conditions of decoupling with protons (Proton Decoupled 
13 C NMR) which induces exaltation of 13 C resonance (Nuclear 
Overhauser Enhancement NOE). The NOE and dipolar 13 C- ! H relaxation 
theory in the proton decoupled 13 C NMR spectra of the macromolecules 
was studied by Doddrell et al. (1971). 



Aromatic 




Fig. 12.13. Fourier transform 1 H NMR spectrum obtained at 270 MHz by Wilson 
et al. (1978) on humic materials extracted by a 0.1 mol (NaOH) L" 1 
solution on a silty potter's clay from Wakanui. A pulsation of 10 |is with 
an inclination angle of 85° was used to visualize a spectral window 
of 3.6 kHz in 8 ko of data points. 



The use of these techniques for soil organic materials was initially 
unsuccessful (Schnitzer and Neyroud 1974). Subsequently several authors 
tried to improve the technique, and Newman et al. (1980) achieved 
noticeable improvement in the quality of the spectra (Fig. 12.14) by 
optimizing the operating conditions, particularly the spacing between the 
radiation pulsations. The optimum interval of 0.2 s also corresponded to 
the acquisition time of the relaxation signals coming from 2 10 5 90° 
impulses of 23 |LlS. The spectra were obtained on an Varian FT-80A 
apparatus operating at 20 MHz for 13 C with proton decoupling at 80 
MHz. Acquisition required collection of 4,000 (coal samples) to 136,000 
(soil humic acid samples) free induction decay spectra then multiplication 
by a filter with a 20 ms time constant, before the Fourier transform. The 
samples were prepared for analysis by suspension of 300 mg of humic 



Characterization of Humic Compounds 



429 



acid in 2 cm 3 of 0.5 mol (NaOH) L" 1 solution for 1 day at 20°C followed 
by ultracentrifugation (84,500 g at 4°C). After dilution to 50% with D 2 0, 
RM" 13 C was measured in tubes with a diameter of 10 mm. 



ch 3 oh 



HCH 2 > n - 



CHOH 




200 



100 
(5/p.p.r 



13, 



Fig. 12.14. Proton decoupled C NMR spectrum of a humic acid in solution ob- 
tained by Newman et al. (1980) with optimized acquisition parameters 
(see conditions described in the text) 



Newman and Tate (1984) used very similar conditions to those 
described above to characterize the humic substances of alkaline extracts 
of soils, the total time needed for spectral acquisition ranged from 10 to 
50 h. 

Preston and Schnitzer (1984) studied the effect of the type of 
extraction (acid or alkaline extraction) and of chemical modifications in 
the extracted material (methylation, hydrolysis using 6 mol (HO) L" 1 
followed or not by methylation) on 13 C NMR spectra of humic materials 
from four types of soils. The materials were dissolved in deuterated 
solvents (CDC1 3 for methylated materials, 0.5 mol (NaOD) L" 1 (D 2 0) 
solution for non-methylated materials). The chemical shifts were mea- 
sured compared to the sodium 3-trimethylsilylpropionate (TSP) for the 
heavy water (D 2 0) solutions and with tetramethylsilane (TMS) for the 
deuterated chloroform (CDC1 3 ) solutions. The spectra were obtained on a 
Bruker WM 250 spectrometer on 10 mm sample tubes with an inter- 
ruption technique of ! H decoupling except during the acquisition time 
(inverse-gated decoupling of Freeman et al. 1972). Some of spectra were 
comparable with those shown in Figure 12.14. 



430 Organic Analysis 

Study by Solid State 13 C NMR 

The extraction of organic matter (cf. Sect. 11.2.1 and 11.3.1 of Chap. 11) 
can result in molecular modifications, particularly if strong bases are 
used as extraction reagents. Moreover, most materials remain in a non- 
extractable state in the humin residue. The in situ study of organic matter 
allows these disadvantages to be overcome. 

However, conventional NMR spectroscopy applied to whole soil 
resulted in only broad and diffuse signals: many dipole-dipole interactions 
produced signals comprising information on the chemical shifts that was 
not clear, and moreover, the spin-lattice relaxation times (Tl) needed too 
long to accumulate the free induction decay signals required to obtain 
quantitative information. The theoretical advance in NMR technique 
(Pines et al. 1973) subsequently made it possible to overcome the 
problem and envisage new developments. In the cross polarization or CP- 
13 C NMR technique, the protons are uncoupled from the 13 C nucleus and 
used to increase the relaxation kinetics of the 13 C nucleus. The signal 
peak widths can be reduced to a degree where the functional groups can 
be partially identified. 

However, the detection of the carbon forms in the whole soil using the 
CP- 13 C NMR method required very organic soils (6% of C according to 
Wilson 1981). This limit has gradually been reduced thanks to 
improvements in the quality of the instruments and also the preliminary 
use of physical fractionation methods such as those described in Chap. 9, 
spectral analysis being limited to the most organic fractions (Barron et al. 
1980). 

The magic angle spinning (MAS) NMR technique, which was 
originally described by Lowe (1959) and subsequently applied to 
polymers by Schaefer and Stejskal (1976), can also help get round 
problems like spectral resolution, and obtain spectra directly on the soils; 
in this technique, the sample is put in fast rotation at an axial slope of 
54°44', which means anisotropic effects can be reduced and isotropic 
shifts can be selected. This technique is often used together with cross 
polarization, and is then known as the CPMAS- 13 C NMR method. 

For soil studies, most authors preferred to apply NMR techniques to 
previously concentrated humic materials, usually humic or fulvic acids. A 
study by Newman et al. (1980) clearly showed the difference in 
resolution that still existed between the 13 C NMR techniques in solution 
and CP 13 C NMR on the solid phase (Fig. 12.15a). 



Characterization of Humic Compounds 431 

Thanks to improvements in this technique, the spectra of CPMAS- 13 C 
NMR obtained by Gerasimowicz and Byler (1985) on humic substances 
showed a better resolution (Fig. 12.15b) than that of Newman et al. (1980). 
Friind and Liidemann (1989) performed instructive comparisons between 
the technique in solution and CPMAS- 13 C NMR which showed that the 
degree of detail provided by the second technique was similar to that of 
the first; in addition, the spectra obtained on a rendzina soil (4.6% of 
carbon) and on its humic extracts and humin residue were compared 
under satisfactory conditions (Fig. 12.15c). The two techniques (liquid phase 
NMR and solid phase CPMAS- 13 C NMR) were recommended by Conte 
et al. (1997a) for the study of organic materials of the soils. State-of-the- 
art CPMAS- 13 C NMR allows observation of organic materials in their 
environment (without fractionation) when the soils are not too low in 
carbon, as attempted by Kinchesh et al. (1995) on Rothamsted soils (UK). 
Other studies such as that of Conte et al. (1997b) on volcanic soils used 
CPMAS- 13 C NMR on extracted humic substances. 

3.4.5 Quantification of Observations by NMR 

Different techniques exist for the quantification of the information 
provided by the NMR signals of humic substances. The oldest derive 
from the study of coal and coal-like materials. 

The method of Brown and Ladner (1960) enables estimation of the 
aromaticity of these carbonaceous materials using the l H NMR spectrum. 
Wilson (1981) proposed an adaptation of this method for use on humic 
materials. 

The rate of aromaticity / a was most often studied by quantification of 
13 C NMR signals. However, techniques for the direct quantification of the 
different peaks of the spectra should be used with caution. The 13 C atoms 
of the different functional groups have different nuclei relaxation times, 
the nuclei with the shortest times making a weaker contribution to the 
total spectrum than those with the longest time. In addition, due to proton 
decoupling, NOE results in an increase in the signal that differs with the 
nature of the nucleus (Wilson 1981). However, these two factors were 
sometimes shown to have no significant influence on the direct 
measurement of the rate of aromaticity (Newman et al. 1980). 



432 



Organic Analysis 



Mud HA 



Aromatic 
C 




Humin 

Native soil 
Washing water -. 



100 
5 ppm 



200 



100 
5 ppm 



13r 



Fig. 12.15 Improvements in solid phase C NMR techniques: (a) CP C NMR 
spectra obtained by Newman et al. (1980) on solid coal, a solid humic 
acid (HA), and a solution of HA, (b) CPMAS- 13 C NMR spectra 
obtained by Gerasimowicz and Byler(1985) on HA from muds at 
different stages of composting treatments (C) CPMAS- 13 C NMR 
spectra obtained by Frbd and Ludem ann (1989) on a rendzina soil, 
on its fulvic (FA) and humic (HA) extracts and on the humin residue 



Characterization of Humic Compounds 433 

Frund and Ludeman (1989) improved quantitative analysis of humic 
materials. Their method enabled simultaneous measurements of 
carboxylic-, aromatic-, carbohydrate- and aliphatic-C rates. Smernik and 
Oades (2000a) highlighted the effect of paramagnetic impurities and of 
purifications by HF treatment (Smernik and Oades, 2000b). Smernik and 
Oades (2003) then Moser and Lefebvre (2004) explored new ways to 
improve 13 C NMR quantification on soil organic matter. Conte et al. 
(2004) reviewed state-of-the-art CPMAS C- 13 -NMR spectroscopy 
applied to natural organic matter. 

12.3.5 Fluorescence Spectroscopy 

Although less widely used than the visible UV (cf. Sect. 12.2.3) or infra- 
red (cf Sect. 12.2.4) absorption spectrometry, fluorescence spectrometry 
has been tested by several authors as a complementary technique to 
characterize humic substances. 

Levesque (1972) used this technique on Fe and P humic complexes. 
The emission spectrum of a fulvic acid has a main peak with a not very 
variable wavelength that moves from 500 to 520 nm when the excitation 
wavelength moves from 400 to 468 nm. As emission spectra generally 
provide little information, excitation spectra were used instead. The 
emission wavelength was then fixed at around 500-520 nm and 
excitation varied in a continuous way between 250 and 500 nm. 

Ghosh and Schnitzer (1980a) observed on their humic substances two 
quite distinct excitation bands, at 465 and 360 nm, and a decrease in 
intensity of the fluorescence with an increase in the ionic force and a 
reduction in the pH; their data were subsequently used to calculate the 
constant of dissociation of humic acids (Goldberg et al. 1987). 

Bachelier (1981) refined these observations with a precise description 
of the excitation spectra on a larger number of soils; he noted the 
presence of seven peaks (including two rare ones) corresponding to slope 
changes on the large 465 nm peak and of two peaks (including one rare 
one) on the large 360 nm peak. 

Bachelier also studied the fluorescence of several types of humic acids 
split on G25 Sephadex gel (1) humic acids of high molecular weight 
which are eluted at the head of the column (coarse humic acid cHA) give 
a weak fluorescent signal; (2) the following band of less condensed 
brown-yellow humic acids gives bright yellow fluorescence under UV 
radiation (fluorescent humic acids f HA); (3) lower fluorescence compounds 
called higher humic acids (hHA) are sometimes found after this band 
and (4) intermediate humic acids (iHA) are sometimes found 



434 



Organic Analysis 



between the two cHA and fHA bands. Figure 12.16 shows some 
excitation spectra obtained on different humic products. Bachelier's 
observations confirm those of Levesque on the low fluorescence of the 
humic complexes with high molecular weight. 




Separation of humic acids 
by gel permeation chromatography 

Higher HA 
Fluorescent HA j: 
Intermediate HA 

Coarse HA 



Vertisol 



Ferrallitic 
soil 




Calcareous 

brown 
soil 



300 



400 



500 excitation 



300 



400 



500 excitation 



Fig. 12.16. Fluorescence excitation spectra of humic materials observed in the 
509-515 nm emission band (Bachelier, 1981): (FA) fulvic acid of 
various samples of soils, (HA) different fractions of humic acids 
separated by gel chromatography (see fractionation diagram above 
the graph) 



Characterization of Humic Compounds 435 

Bachelier (1984) used fluorescence spectrometry to distinguish the 
degrees of condensation of humic acids. Nayak et al. (1985) studied the 
fluorescence of humic acids in different solvents. The fluorescence 
polarization technique enabled further information to be obtained on the 
aggregation and conformation of humic molecules and was used to this 
end in a study of fulvic acid by Lapen and Seitz (1982). 

12.3.6 Electron Spin Resonance (ESR) Spectroscopy 

When molecules containing unpaired electrons are placed in a magnetic 
field, interaction occurs between the magnetic moment of these electrons 
and the applied field which results in decoupling into two discrete states 
of energy of each unpaired electron. This is electron spin resonance 
(ESR) sometimes still called electron paramagnetic resonance (EPR). 
ESR spectroscopy uses electromagnetic excitation radiation located in the 
spectral field of microwaves; it is used for the study of compounds 
containing unpaired electrons, primarily free radicals. 

The energy of an unpaired electron in a magnetic field is given by 

E = -g{3M z H , 
where G represents the spectroscopic split factor which has a value of 
2.0023 for a free electron, /?the magneton of Bohr, M z the component of 
the angular spin moment in the direction of the z-axis of the applied 
magnetic field which can take the discrete values +1/2 and -1/2, H the 
force of the magnetic field. For a given value of H the difference in 
energy AE between two states of discrete spin of the electron is 

AE = g/3H 
the phenomenon of resonance occurs when this energy is equal to that of 
the applied field: 

AE = hv 
and H being Planck's constant and vthe frequency. 

In his pioneer work, Rex (1960) used ESR spectroscopy to highlight 
the radical nature of humic molecules. MacCarthy and Rice (1985) listed 
several works reporting the use of ESR spectroscopy for humic 
substances. The spectra of humic substances are generally simple signals 
identified by their position and width (Fig. 12.17). The hyperfine lines 
that can be identified in some molecules are not usually present in humic 
spectra. 



436 



Organic Analysis 




-10 -5 o +5 +10 

Scanning range (Gauss) 

Fig. 12.17 Electron spin resonance spectrum of a fulic acid (Schnitzer and 
Skinner, 1969) 

Schnitzer and Skinner (1969) showed on ten humic materials that the 
most variable parameter deduced from these spectra is the spin 
concentration. This is determined by comparison with a standard of 
calibration, the number of radicals being proportional to the signal 
surface. In humic substances, the concentration is found around 10 18 spins 
g 1 . Schnitzer and Skinner also tested the influence of several factors on 
the spin concentrations of humic substances (chemical modifications, 
heating) as well as the relation between other parameters and the spin 
concentration (molecular weight, E4:E6 ratio, number of molecules of a 
given weight per free radical). Riffaldi and Schnitzer (1972) studied the 
effect of the experimental conditions on the ESR spectra of humic 
substances to highlight mistakes due to too rapid interpretation of these 
spectra. MacCarty and Rice (1985) also commented on the relative 
poverty of the information from ESR spectrometry for the study from the 
humic substance. However, subsequent technological developments 
changed this situation. For example, Senesi et al. (1989) provided more 
detailed spectra than previous authors and Senesi (1990) reviewed state- 
of-the-art and potential development of ESR technique in its application 
to soil chemistry. Saab and Martin-Neto (2004) studied semiquinone free 
radicals of humic substances by ESR. 



Characterization of Humic Compounds 437 

12.3.7 Measurements of molecular weight and molecular size 
Principles 

Measurement of molecular weight is a traditional procedure in structural 
chemistry. As a complement to ultimate analysis, the molecular weight 
provides an empirical formula for the compound concerned before the 
determination of the structural formula (functional groups) using 
spectroscopic and chemical methods. The measurement of molecular 
weight and size has been attempted many times on humic substances and 
several reviews have been devoted to the subject (e.g. Orlov et al. 1975; 
Stevenson 1982; Wershaw and Aiken 1985; Buurman 2001). It has also 
been used to measure the humic substances of water (Yu-Ping Chin et al. 
1994, Yamada et al. 2000) or atmosphere (Samburova et al. 2005) 
However, the results differed considerably firstly depending on the 
technique used, and secondly because, as emphasised by the authors of 
these reviews, humic substances are not discrete chemical entities but 
complex mixtures of polydisperse organic substances with a wide range 
of molecular size. 

The problem with measuring the molecular weight of mixtures 
nevertheless was studied during many years. In 1935 Lansing and 
Kraemer pointed out that the most commonly used methods resulted in 
average molecular weights and that, depending on the method of 
measurement used, these average weights could not always be compared. 
Three types of average molecular weights corresponding to three types of 
measurement are now used: 

Number-Average Molecular Weight M n 

This is obtained using methods that measure the number of molecules 
(generally in a diluted solution) irrespective of their size. It is expressed 
by: 

M n = Zw.M./Z n., 

i i r 

where rif is the number of molecules of molecular weight Mi M n is 
determined by all measurements corresponding to a thermodynamic 
property connected to a number of molecules in solution (colligative 



438 Organic Analysis 

property): lowering of vapour pressure, lowering of the freezing point, 
rise in the boiling point (Raoult laws), osmotic pressure. 

Weight-Average Molecular Weight M w 

Weight-average molecular weight is measured by methods concerned 
with the masses of different materials, like light scattering and ultra- 
centrifuge sedimentation; it is expressed by: 

M w = ZwiMVZ wt = X rif Mt 2 /Z m Mi, 

where w. represents the mass fraction of each species; 

The z- Average Molecular Weight M z 

This can also be calculated with the ultracentrifugation data obtained and 
is expressed by: 

M z = X wt Mi 2 /S wt Mi = X m Mi 3 /L m Mi 2 . 

In a monodisperse system M n = M w = M z but this is not the case in 
poly disperse systems; the number-average molecular weight then tends to 
represent the lowest molecular weights whereas M w tends to represent 
the heaviest particles of the mixture (Wershaw and Aiken, 1985). Orlov 
et al. (1975) expected M w to be better correlated with the known 
properties of humic substances. In heterogeneous systems, M z > M w > 
M n , the M w :M n ratio can generally be used to calculate the degree of 
polydispersity (Stevenson 1982). 

In addition to methods for the measurement of the average molecular 
weight, another group of methods measures the size of the molecules, e.g. 
gel filtration, ultrafiltration and small angle X-ray scattering. In these 
methods, model compounds of known molecular weight and composition 
are used to determine the molecular weight of humic substances, but 
problems can occur if these compounds are too different from the humic 
molecules (Wershaw and Aiken 1985). 

Methods for the Measurement of Molecular Size 



Gel Exclusion Chromatography 

The methods of gel exclusion (or gel permeation) chromatography are 
described in Sect. 12.2.1 and 12.3.1. It should be noted that Reuter and 
Perdue (1981) found a very big difference between the expected 
molecular weights on humic fractions of Sephadex gel and the number- 
average molecular weights actually measured. Plechanov (1983) used the 



Characterization of Humic Compounds 439 

Sephadex LH60/dimethylformamide/acetic acid system for the measurement 
of the molecular weight of humic substances. Nobili et al. (1989) reviewed 
the use of gel chromatography for the measurement of the molecular size 
of humic substances. 

Ultrafiltration 

Ultrafiltration by pressure filtration through a membrane is also used for 
the separation of macromolecules as a function of their molecular size. 
This technique is similar to reverse osmosis, except with respect to the 
size of the particles that can be split: reverse osmosis separates the 
particles of molecular size near to those of the solvent whereas 
ultrafiltration separates particles approximately 10 times the size of the 
solvent i.e. up to 0.5 |Lim (Wershaw and Aiken 1985). A large number of 
different types of membranes exist which are classified by their 
manufacturers according to their threshold of cut expressed in molecular 
weight. Nevertheless ultrafiltration is not a technique for separation by 
weight but by molecular size. A review by Wershaw and Aiken (1985) 
provided a lot of useful information about this technique. 

Scattering of Electromagnetic Radiation 

The principle of this technique for light scattering was described by 
Kerker and Milton (1968) and by Guinier and Fournet (1955) for small 
angle X-ray scattering. The reader is advised to consult these publications 
for a comprehensive explanation of the phenomenon and to refer to 
Wershaw and Aiken (1985) and Wershaw (1989) for the use of this 
technique for humic substances. 

Methods of Measurement of Molecular Weight 

Determination of the Number-Average Molecular Weight by 
Measurement of Colligative Properties 

By definition, a colligative property is a thermodynamic property which 
depends on the number of particles in a solution independent of their 
nature. At the infinite dilution limit, each one of these properties is 
proportional to the number of molecules of solute present in the solution. 
The classical theory of each colligative property is described found in all 
chemistry and physics handbooks. The most widely used techniques for 
the measurement of molecular weight of humic substances are cryoscopy 
and vapour pressure osmometry. Cryoscopy records the drop in the 
temperature during solidification of the solvent in the presence of the 
solute to be studied. Vapour pressure osmometry measures the change in 
osmotic pressure resulting from the passage of a solvent through a 



440 Organic Analysis 

membrane from a diluted solution to more concentrated one. For a 
description of these methods and their use for humic substances, see 
Stevenson (1982), Wershaw and Aiken (1985) and Aiken and Gillam 
(1989). 

Ultracentrifugation 

There are two distinct groups of ultracentrifugation techniques, those 
concerned with sedimentation kinetics, and those concerned with 
ultracentrifugation equilibrium. The first group was the most commonly 
used on humic substances (Wershaw and Aiken 1985) sometimes as a 
complement to other fractionation methods (Cameron et al. 1972b), 
although the centrifugation equilibrium provides a wealth of information 
since M n , M w and M z can be determined at the same time (Posner and 
Creeth 1972). The theory and implementation of the first technique is 
detailed in Cameron et al. (1972b), the second by Posner and Creeth 
(1972), and a review by Swift (1989) gives a detailed description of all 
ultracentrifugation techniques. 

Viscosimetry 

Measurements of viscosity can provide significant information 
concerning the size and the shape of the molecules. The well-known 
Oswald viscometer records the times of passage of the solution and 
solvent between two reference points marked on the apparatus. The 
molecular weight can be estimated from measurements of viscosity using 
the Staudinger equation: [77] = kM a , in which intrinsic viscosity 7] is 
linked to molecular weight M by two adjustable parameters (Ghosh and 
Schnitzer, 1980b). Techniques based on measurement of viscosity were 
reviewed by Clapp et al. (1989). 

Flow FFF (cf. Sect. 12.3.1) was also once considered to be promising 
for the measurement of molecular weight of humic substances (Beckett 
etal. 1987). 

12.3.8 Microscopic Observations 

Several studies report observations of humic materials using optical 
microscopy, transmission electron microscopy and scanning electron 
microscopy. The first difficulty is linked with the different ways the 
sample can be modified depending on the preparation techniques (e.g. the 
degree of separation of inorganic materials, molecular modifications with 
respect to the ionic force and pH), and the second difficulty is the 
conditions of the observation itself (heating of the sample). Bachelier 
(1983) carried out observations on nine different types of soil using three 



Characterization of Humic Compounds 441 

techniques: frozen humic acid solutions under a binocular magnifying 
glass, desiccated humic acid solutions under a transmission electron 
microscope, humic acid solutions freeze-dried on gilt aluminium film 
under a scanning electron microscope. The latter requires metallization of 
the non-conducting substances before observation. 

Chen and Schnitzer (1976) studied the influence of pH on the 
appearance of humic acids by scanning electron microscopy while 
Stevenson and Schnitzer (1982) studied the same effect by transmission 
electron microscopy. Chen and Schnitzer (1976) used transmission 
electron microscopy for the study of metallic complexes of fulvic acids. 

Scanning electron microscopy was used by Chen et al. (1978) for the 
comparison of humic acids from Mediterranean soils. Tan (1985) 
provided detailed methodological information particularly concerning the 
preparation of the samples. 

12.3.9 Other Techniques 

The main techniques used for structural characterization are described in 
this chapter. However other techniques can also be used to improve our 
knowledge of the structures of humic compounds and their linkage with 
mineral materials. 

X-ray techniques are not limited to small angle X-ray scattering 
described in "Scattering of Electromagnetic Radiation" in Sect. 12.3.7; X- 
ray diffraction was also used by Schnitzer (1978), and can provide useful 
information in spite of the non-crystalline character of humic substances. 

As far as electrochemical methods are concerned, a study of humic 
acid characterization by polarography was described by Shinozuka and 
Hayano(1987). 

The use of FTIR spectroscopy makes it possible to increase the 
resolution and to decrease the background noise of the IR spectra. 
However, these advantages may not be apparent in the study of humic 
substances because of their molecular nature (MacCarthy and Rice, 
1985). 

More recent techniques like X-ray photoelectron spectroscopy (XPS) 
and Mossbauer spectroscopy have not been widely used for the study of 
humic substances. XPS, also called electron spectroscopy for chemical 
analysis (ESCA), can only be used with solid materials because it 
requires a high vacuum; it is based on the analysis of the electrons 
emitted by the internal electron-shells atoms when they are subjected to 
X-ray bombardment of sufficient energy; Defosse and Rouxhet (1980) 
introduced this technique in soil analysis. 



442 Organic Analysis 

Mossbauer spectroscopy is not directly useable for the study of the 
structure of metal compounds, but can be used for the study of 
organometallic bonds, and particularly for the study of complexes of 
iron-humic compounds (Goodman and Cheshire, 1979). 

It will be interesting to see whether future progress results from new 
technologies or from synthetic studies on molecular models as was the 
case for the identification of the double helix of ADN by Watson and 
Creek. 



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Spectrometric Characterizations 



UV-Visible, IR, Fluorescence, ESR Spectrometries 

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Rex RW (1960) Electron paramagnetic resonance studies on stable free radicals 

in lignins and humic acids. Nature, 188, 1 185-1 186 
Riffaldi R and Schnitzer M (1972) Effects of divers experimental conditions on 

ESR spectra of humic substances. Geoderma, 8, 1-10 
Russel JD and Anderson HA (1977) Comment on "spectroscopic infra-rouge de 

quelques fractions d' acides humiques obtenues sur Sephadex" Plant 

Soil, 48, 547-548 



Characterization of Humic Compounds 447 

Schnitzer M and Skinner SIM (1969) Free radicals in soil humic compounds. 

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Saab SC and Martin-Neto L (2004) Studies of Semiquinone Free Radicals by 

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Sci, 14, 77-129 

Nuclear Magnetic Resonance 

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Brown JK and Ladner WR (1960) A study of the hydrogen distribution in coal- 
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Quantitative differences by liquid- and solid-state 13 C NMR 
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Conte P, Piccolo A, van Lagen B, Buurman P and de Jager PA (1997b) 
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Nuclear Magnetic Resonance Spectroscopy, 44, 2 1 5-223 

Doddrell D, Glushko V and Allerhand A (1972) Theory of the Nuclear 
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Frund R. and Liidemann HD (1989) The quantitative analysis of solution and 
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Kinchesh P, Powlson DS and Randall EW (1995) 13 C NMR studies of organic 
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Lentz H, Ludemann HD and Ziechmann W (1977) Proton resonance spectra of 
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Lowe IJ (1959) Free induction decay of rotating solids. Phys. Rev. Lett., 2, 
285-287 



448 Organic Analysis 

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13 C-NMR. J. Soil Sci, 31, 623-631 
Pines A, Gibby MG and Waugh JS (1973) Proton enhanced NMR of dilute spins 

in solids. J. Chem. Phys., 59, 569-590 
Preston CM and Schnitzer M (1984) Effects of chemical modifications and 

extractants on the 13 C NMR spectra of humic materials. Soil Sci. Soc. 

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Schaefer J and Stejskal EO (1976) C-13 NMR of polymers spinning at the magic 

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Schnitzer M and Barton DHR (1963) A new approach to the humic acid 

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Schnitzer M and Neyroud JA (1974) The chemistry of high molecular weight 

fulvic acid fractions. Can. J. Chem., 52, 4123-4132 
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Characterization of Humic Compounds 449 

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Methods of Characterization by Fragmentation 

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359 
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Cheshire MV, Cranwell PA and Haworth RD (1968) Humic acid - III. 

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267 
Griffith SM and Schnitzer M (1976) The alkaline cupric oxide oxidation of 

humic and fulvic acids extracted from tropical volcanic soils, Soil Sci., 

122, 191-201 
Hansen EH and Schnitzer M (1967) Nitric acid oxidation of Danish illuvial 

organic matter. Soil Sci. Soc. Am. Proc, 31, 79-85 
Hansen EH and Schnitzer M (1969) Zn-dust distillation and fusion of a soil 

Humic and fulvic acid. Soil Sci. Soc. Am. Proceed., 33, 29 
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450 Organic Analysis 

Jackson MP, Swift RS, Posner AM, Knox JR (1972) Phenolic degradation of 

humic acid. Soil Sci., 114, 75-78 
Kahn SU and Schnitzer M (1971a) The permanganate oxidation of methylated 

and unmethylated humic acids extracted from Solonetz, Solod and 

Chernozem Ah horizons. Israel J. Chem., 9, 667-677 
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fulvic acid, a soil humic fraction. Can. J. Chem., 49, 2302-2309 
Kahn SU and Schnitzer M (1972a) Permanganate oxidation of humic acids, 

fulvic acids, and humins extracted from Ah horizons of a black 

chernozem, a black solod and a black solonetz soil. Can. J. Soil Sci., 

52,43-51 
Kahn SU and Schnitzer M (1972b) Permanganate oxidation of humic acids 

extracted from a GrayGrey wooded soil under different cropping 

systems and fertilizer treatments. Geoderma, 7, 1 13-120 
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organic matter. 1 .Introduction and methodology, Geoderma, 4, 47-55 
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sodium amalgam. Soil Sci., 102, 85 
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of pyrolysis mass spectra from melanins of soil fungi, model phenolic 

polymers and humic acids from soil, peat and composted straw. 

Geoderma, 17, 239-252 
Meuzelaar HLC, Posthumus MA, Kistemaker PG and Kistemaker J (1973) Curie 

point pyrolysis in direct combination with low voltage electron impact 

ionization spectrometry. Anal. Chem., 45, 1546-1549 
Morrison RI (1963) Products of the alkaline nitrobenzene oxidation of soil 

organic matter. J. Soil Sci., 14, 2 
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oxidation of humic and fulvic acid, Soil Sci. Soc. Am. Proc, 38, 907- 

913 
Neyroud JA and Schnitzer M (1975) The alkaline hydrolysis of humic 

substances. Geoderma, 13, 171-188 
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lignin, Soil Sci., 116, 93-100 
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II). Soil Biol. Biochem., 4, 513-531 



Characterization of Humic Compounds 451 

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Schnitzer M and Skinner SIM (1974) The peracetic acid oxidation of humic 
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acids. Soil Set, 103,383 

Stevenson FJ (1989) Reductive cleavage of humic substances. In : Humic 
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Malcolm RL and Swift RS ed., Wiley New York, 122-142 

Other methods (microscopy, X-ray, electrochemistry, etc.) 

Bachelier G (1983) Figures de dessication des acides humiques, Rapport multig. 

ORSTOM, 14 p 
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and of a fulvic acid and its metal and clay complexes. Soil Sci. Soc. Am. 

J., 40, 682-686 
Chen Y, Senesi N and Schnitzer M (1976) Chemical and physical characteristics 

of humic and fulvic acids extracted from soils of the mediterranean 

region. Geoderma, 20, 87-104 
Shinozuka N and Hayano S (1987) Polarographic characterization of humic acid. 

Soil Sci., 143, 157-161 
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extracted humic and fulvic acids, Soil Sci., 133, 334-345 
Tan KH (1985) Scanning electron microscopy of humic matter as influenced by 

methods of preparation. Soil Sci. Soc. Am. J., 49, 1 185-1 191 
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media. J. Soil Sci., 30, 85-91 



13 



Measurement of Non-Humic Molecules 



13.1 Introduction 



13.1.1 Non-Humic Molecules 

Soil organic matter probably contains the majority of biochemical 
compounds synthesized by living organisms (Stevenson 1982). In addition 
to humic molecules, whose quantification (described in Chap. 11) is 
technically simpler than the structural characterization of molecules (cf 
Chap. 12), a large number of other molecules of known structure are also 
present in soils. The most abundant can be classified in three main 
groups: 

1. Nitrogenous molecules, which are often studied by their fragmentation 
products during acid hydrolysis. 

2. Polysaccharides, mostly not nitrogenous molecules, which are also 
often studied by their fragmentation products. 

3. The lipid fraction, which are sometimes called soil bitumens and 
contain a range of molecules extractable with the solvents used for 
fats; this fraction also contains many of the organic pollutants and 
xenobiotic residues that can contaminate the soil. 

Titration techniques for (1) nitrogenous molecules are described in 
Chap. 14. This chapter describes the main titration techniques for the 
second and third types of molecules. 

13.1.2 Soil Carbohydrates 



Total Composition 

Sugars (also improperly called carbohydrates because of an empirical 
formula corresponding to C„(H 2 0)„) account for 5-25% of soil organic 



454 Mineralogical Analysis 

matter. Aside from traces of free sugars which can be extracted from the 
soil by water, soil carbohydrates are components of polysaccharides. It is 
practically impossible to isolate soluble fractions of polysaccharides for 
identification (Cheshire 1979). Consequently it is difficult to know if 
sugars come from a heterogeneous mixture of polysaccharides or from a 
single particularly complex polysaccharide. 

Eight neutral carbohydrates can be identified by hydrolysis of soils. 
These can be classified in three main groups: hexose, deoxyhexose and 
pentose sugars. Two acid sugars (uronic acids) and two basic sugars 
(hexosamines) can also be identified. 

Ranked in descending order, hexose sugars account for from 
12-4% of organic matter and include glucose, galactose and mannose. 
Uronic acids account for 1-5% of soil organic matter and contain roughly 
equal parts of galacturonic and glucoronic acids. Pentose sugars are 
present in smaller quantities and contain mainly arabinose and xylose as 
well as traces of ribose. Fucose and rhamnose deoxyhexoses are found in 
similar concentrations to pentose sugars. 

Amino sugars (cf. Chap. 14) are present still in lower concentrations in 
the soil; their chief components are hexosamines in the form of 
galactosamine and glucosamine. 

Traces of other sugars can also be found in the soil: four methyl 
sugars, two alcohol sugars (inositol and manitol), two hexose sugars 
(fructose and sorbose), a pentose sugar (deoxyribose) and a hexosamine 
sugar (N- acetyl glucosamine). 

Sugars and Types of Soils 

Many authors have tried to characterize soils by quantification of the 
sugars that result from acid hydrolysis of polysaccharides. Folsom et al. 
(1974) found an almost linear relationship between total carbohydrate 
content and soil organic carbon content; however a curvature appeared 
for the range of very organic horizons which contained a lower 
proportion of sugars. These authors reported that grassland soils 
contained more pentoses and less hexoses than forest soils and also that 
the proportion of mannose increased with soil depth, indicating greater 
stability of this sugar. 

Singhal and Sharma (1985) reported that total carbohydrates and 
organic carbon contents of forest soils varied in the same way whatever 
the tree cover. They observed no difference in the relative proportions of 
these sugars. MacGrath (1973) also found a very constant value for the 
relative composition of sugars in 38 Irish grassland soils. 



Non-humic Molecules 455 

Cheshire and Anderson (1975) observed a higher quantity of total 
sugar in cultivated soils than in uncultivated soils but the relative 
proportions were the same. 

Distribution and Origin 

Another aspect of the problem is linked to the distribution of 
polysaccharides in the fractions of soil organic matter. Diluted alkaline 
solutions are the best reagent for polysaccharide extraction although the 
majority of the polysaccharides remain associated with humin residues. 
Acidification of alkaline extracts precipitates humic acids and the 
majority of the polysaccharides remain in the acid soluble fraction of 
fulvic acid (Cheshire 1979; Barriuso et al. 1985). Bagautdinov et al. 
(1984) separated one fraction from a fulvic acid solution which contained 
mainly polysaccharides with a molecular weight of 27,000-28,000. 

Many authors have tried to determine whether soil sugars are of 
microbial or plant origin. This is not a simple task since none of the main 
sugars can be classified exclusively as being of plant or microbial origin 
(Cheshire 1979). There are more similarities than differences in sugars 
between humic acids and fungi melanins (Coelho et al. 1988). 

Incubation experiments using labelled glucose led to labelling of all 
sugars and, to a lesser extent, of amino acids. Deoxyhexoses have been 
identified as the most stable synthesized sugars (Cheshire 1979). With an 
increase in incubation time, an increasingly large proportion of labelled 
carbon was found in humin (Guckert et al. 1971). Hexose sugars are the 
main sugars synthesized by soil micro-organisms (Oades 1974). 

Francois (1988) and Murayama (1983, 1984, 1988) reported that they 
had been unable to identify a specific origin for glucose, galactose and 
ribose but that xylose and arabinose are primarily of plant origin, while 
rhamnose, mannose and fucose are very often synthesized by micro- 
organisms but are also present in root exudates of various plants. 

Frangois (1988) showed that total sugars are primarily concentrated in 
fresh roots and in the 5-25 |Lim soil particle fraction. A marked relative 
reduction in xylose content and an increase in mannose and rhamnose 
contents were measured in the finer fractions. Feller (1991) used the 
xylose:mannose ratio as an indicator of the microbial or plant origin of 
the organic matter. This ratio had the lowest value (0.5-2) in the organic 
matter of the clay-organic compartment <2 |Lim; its value ranged between 
1 and 3 in the 2-20 |Lim fraction and between 5 and 10 in the >20 |Lim 
fraction (cf. Chap. 9). 



456 Mineralogical Analysis 

Principle of Titration 

Free sugars can be titrated on aqueous extracts of soils. Polysaccharide 
titration has three main stages: 

- Acid hydrolysis of polysaccharides; 

- Possible purification of the hydro lysate; and 

- Titration of the hydro lysate. 

Titration of free or hydrolysed sugars uses two types of techniques: 

- Global colorimetric methods for reducing sugars and 

- Chromatographic methods allowing the measurement of each indivi- 
dual sugar. 

13.1.3 Soil Lipids 

Soil lipids are rather complex mixtures of compounds one of whose 
common characteristics is solubility in a range of organic solvents or 
mixtures of solvents. This fraction includes groups such as free fatty 
acids, hydrocarbons, polar or non-polar lipids, steroids, waxes and resins. 

The majority of lipids can be classified in three main groups: fats, 
waxes and resins. The resins are the most polar compounds and are thus 
most soluble in methanol and ethanol, and this property can be used to 
separate them. 

Lipids are present in the soil in smaller quantities than nitrogenous 
compounds and polysaccharides. According to Stevenson (1982) they 
account for 2-6% of organic matter) and according to Jambu et al. (1978) 
up to 20% in certain soils. Lipids are generally present in higher 
concentrations in acid soils. The most fertile soils are generally poor in 
lipids. The presence of lipids may even be related to the old concept of 
soil sickness (Stevenson 1966) and depend on humus content, soil 
aeration and texture (Jambu et al. 1978). 

The extraction of soil lipids may be complicated by the fact they bond 
to a varying extent with other organic or inorganic compounds in the soil. 
Most pesticides and other organic pollutants of the soils are also extracted 
with the lipid fraction. 



Non-humic Molecules 457 

13.1.4 Pesticides and Pollutants 

Most of the products used in food and agricultural chemistry can be 
found in soils. 

Organic pollutants that result from the breakdown of products in the 
environment can be extremely varied, although the majority belong to the 
following main groups: 

- Poly chlorinated biphenyls (PCBs) are families of chlorinated pollutants 
including about 30 compounds; traces of PCB can be detected in a very 
large number of substrates; 

-Polynuclear aromatic hydrocarbons (PAH) include 15-20 compounds 
(e.g. naphthalene, phenantrene, anthracene or pyrene) that are resistant 
to degradation; and 

- Dioxins are also residues of degradation but are present in more limited 
quantities. 

Pesticides are generally not very stable molecules used in agriculture in 
a wide range of products like insecticides, acaricides, nematocides, 
repellents, fungicides, herbicides and poisons. They comprise a very large 
number of compounds. Although there is a general trend towards 
molecules that are less and less toxic for humans, and more and more 
degradable, there is also an increase in the total amount used as a result of 
gene mutations and of the development of resistance in living organisms. 
Calvet et al. (2005) summarized current knowledge about pesticides and 
their agronomic and environmental consequences for soils. 

More than one thousand compounds are sold as pesticides and these 
are difficult to classify. Some of the main families are: 

- Organochlorinated products including the first historically sold 
synthetic molecules such as DDT, dieldrin or lindane; these products 
are less used today because of their toxicity and low rate of 
degradability; however, they are still found in the environment since 
they accumulated in the lipids of living organisms; nowadays new 
halogenated molecules are used; 

- Organophosphorous products were often used to replace the first 
organochlorinated products; 

- Triazine herbicides; 

- Acid herbicides like chlorophenoxyacetic acids, picloram or dicamba; 

- Carbamates and thiocarbamates, which are often used as systemic 
insecticides; and 

- Pyrethrinoids, which are synthetic molecules derived from natural 
pyrethrins and are commonly used as insecticides because they are not 
exchanged in the blood of human beings or warm-blooded animals. 



458 Mineralogical Analysis 

13.2 Classical Techniques 

13.2.1 Acid Hydrolysis of Polysaccharides 
Principle of the Technique and Main Difficulties 

Hot diluted acids are capable of completely hydro lyzing polysaccharides 
but in most soils they only hydrolyze about 75%. 

To achieve complete hydrolysis of polysaccharides, e.g. cellulose or 
soil polysaccharides, a preliminary treatment with a more concentrated 
acid is required (Cheshire 1979). During hydrolysis with diluted acids, 
release of hexose sugars increases with an increase in the concentration of 
the acid. Alone, diluted acids are generally not appropriate for 
quantitative titration of hexose sugars. 

Pentose sugars are more easily released than hexose sugars but they are also 
more easily destroyed during acid hydrolysis and, depending on the soil, they 
may be more efficiently titrated by hydrolysis in more diluted medium. 

Ivarson and Sowden (1962) were the first to recommend cold 
pretreatment with 12 mol L" 1 sulphuric acid (72%) followed by dilution of 
the acid to 0.5 mol L~ and heating at reflux. In samples of litter, this attack 
released almost three times more hexoses than hydrolysis without 
pretreatment, but the release of pentoses was reduced by approximately 
20% in the case of conifer litter. 

Gupta and Sowden (1965) confirmed on four soils a very positive 
effect of pretreatments for the titration of hexoses and pentoses, except in 
some cases where deoxyhexoses were partly destroyed. 

Cheshire and Mundie (1966) optimized the time of cold pretreatment 
with 12 mol L" 1 sulphuric acid. The sugars measured by orcinol colorimetry 
reached maximum towards 16 h then decreased, whereas those measured 
by anthrone colorimetry (hexoses) continued to increase up to 40 h. The 
length of pretreatment thus influenced the time of attack at reflux with the 
0.5 mol L" 1 sulphuric acid solution necessary for the maximum release of 
sugar. After 16 h cold pretreatment, 5 h of hot attack were sufficient to 
reach maximum, whereas after 2 h pretreatment, nearly 20 h of attack were 
necessary. Finally, these authors concluded that there is no perfect method 
of hydrolysis enabling the complete release of glucose without partially 
destroying pentoses or deoxyhexoses. The 16 h treatment with 12 
mol (H 2 S0 4 ) L" 1 at 20°C followed by heating for 5 h at reflux with 0.5 
mol (H 2 S0 4 ) L" 1 was recommended because in several cases it resulted in 
the highest rate of release of glucose. This reason is not very convincing in 
the case of studies on the origin of sugars in the soil where glucose is not 
representative of a sugar of plant or microbial origin (cf. Sect. 13.1.1). 



Non-humic Molecules 459 

Oades et al. (1970) focussed on hydrolysis conditions for 
chromatographic titration of sugars. For a soil with no plant fragments, 
they found the best extracted sugar contents by direct attack at reflux for 
1 h with a 5 mol ( 1 /2H 2 S0 4 ) L" 1 solution. However, this attack released 
less glucose than another technique similar to that of Cheshire and 
Mundie (16 h in 13 mol (H 2 S0 4 ) L" 1 cold reagent followed by heating at 
reflux for 2 h in 0.5 mol (H 2 S0 4 ) L" 1 ). But Oades' technique released a 
higher quantity of other sugars, particularly xylose, rhamnose and fucose. 
However in a sandy silt soil, there is a risk in significant breakdown of 
sugars when the duration of hydrolysis exceeds 20 min for xylose, 40 min 
for arabinose and about 1 h for other sugars (not the same risk in the 
method with pretreatment). 

In fact for total sugar contents, the results of the method with heating 
at reflux for 20 min in 5 mol (V2H SO ) L 1 were not very different from 
those obtained using cold pretreatment, except for samples rich in 
materials where method with pretreatment is recommended. The Oades' 
method provided more pentoses and deoxyhexoses but much less 
glucose. To release glucose, a longer attack with 5 mol (!/2H 2 S0 4 ) L~ was 
necessary but with the risk of destroying pentose and deoxyhexose 
sugars. To obtain maximum rates for glucose and other sugars 
simultaneously Oades et al. recommended heating at reflux for 20 min 
with 5 mol (^H^O^ L" 1 reagent then filtration followed by 16 h of cold 
maceration in 26 mol (ViR^SO^ L~ reagent and heating at reflux for 5 h 
in 1 mol (!/2H 2 S0 4 ) L" 1 reagent. Initial heating at reflux with 5 
mol (V2H2SO4) L" 1 reagent for only 20 min may be sufficient for not very 
organic soils but there is a risk of underestimating glucose. 

The majority of more recent studies did not continue to optimize 
hydrolysis conditions; Coelho et al. (1988), Murayama (1987), Cheshire 
and Griffiths (1989) used the method of Oades et al. (1970) with 
hydrolysis in two stages. They sometimes distinguished sugars released 
by heating at reflux for 20 min with 5 M H 2 S0 4 , called non-cellulose 
sugars, from sugars released by later hydrolysis (heating at reflux for 5 h 
with 1 N H 2 S0 4 preceded by maceration in 26 N H 2 S0 4 ), called cellulose 
sugars. 

Arschad and Schnitzer (1987) and Baldock et al. (1987) used the 
method developed by Spiteller (1980) which is similar to that of Cheshire 
and Mundie (1966) with respect to the conditions of hydrolysis: 
maceration for 16 h with 26 N H 2 S0 4 followed by boiling at reflux for 5 h 
withNH 2 S0 4 . Singhal and Sharma (1985) used the method of Gupta 
(1967): 2 h of extraction at low temperature with 72% H 2 S0 4 then 



460 Mineralogical Analysis 

heating at reflux for 16 h after dilution. Benzing-Purdie and Nikiforuk 
(1989) used a simpler technique for hydrolysis at 105°C for 18 h in 2 N 
H 2 S0 4 and compared it with the method of Cheshire and Mundie (1966). 
Guckert (1973) also used a similar technique with hydrolysis with 3 
mol (VtH 2 S0 4 ) L" 1 acid at 80°C for 24 h after extraction. 

Without cold pretreatment, with 4.5 h hydrolysis, titration of sugars by 
both anthrone and ferricyanide colorimetry reached maximum with a 5 N 
acid concentration and a temperature of 100°C (in closed flasks) and 
displayed a much less significant influence of the acid concentration than 
of temperature (Pansu 1992). Cold pretreatment increased the quantities 
of sugars released and the optimum temperature for the subsequent attack 
moved to between 70 and 90°C with a weaker acid (1 mol ( 1 /2H 2 S0 4 ) L 1 , 
in agreement with the results of Cheshire and Mundie (1966). 

Pansu (1992) also showed good resistance to the degradation of known 
quantities of monosaccharides added in the attack solutions. Commercial 
crystallized cellulose showed good resistance to concentrated acids at 
105°C whatever the concentration of the sulphuric acid, and even to 
hydrochloric acid. Cellulose hydrolysis only became significant (88%) with 
the method including a cold pretreatment with the 26 mol ( 1 /2H 2 S0 4 ) L" 1 
reagent. Hydrolysis of commercial cellulose released not only glucose (66- 
86% of the total), but also galactose (10-30%), mannose (1-2%), xylose 
(approximately 1%) andribose (approximately 1%). 

Equipment and Reagents 

-Inorganic purified water: demineralized water purified on activated 
carbon (standard Millipore) or water distilled after reflux attack in the 
presence of a little potassium permanganate and sulphuric acid. 

- Six round bottom boiling flasks (100 mL) with ground stopper or six 
flasks with PTFE joint screw stopper (100 mL). 

- Set of six condensors (only for the method with reflux heating). 
-Buchner funnels with standard GF/A glass fibre filters ^ 4 cm 

(Whatman No. 1820 042, or similar). 

- 13 mol L" 1 concentrated sulphuric acid solution: add 278 mL inorganic 
water in a 1 L volumetric flask; carefully add while agitating and 
cooling in a cold water bath, 722 mL of concentrated reference grade 
sulphuric acid; complete to 1 L. 

- 2.5 mol (H 2 S0 4 ) L" 1 (5 N) sulphuric acid solution: proceed as given 
earlier but with 139 mL of acid and complete to 1 L with inorganic 
water. 



Non-humic Molecules 461 

Procedure 

Section 13.2.1 earlier mentions that our current state of knowledge makes 
it difficult to recommend a precise standardized attack that is valid for all 
soils. The conditions of hydrolysis need to be adjusted to the type of 
study required. Based on the review of the literature in Sect. 13.2.1, the 
three alternative procedures listed later can all be used. 

Attack Favouring the Measurement of Pentoses 

Put 5 g of soil and 40 mL of 2.5 mol L" 1 sulphuric acid in a 100 mL round 
bottom boiling flask. Connect to the condensor and heat at reflux for 20 
min (alternatively heat in a flask with a screw cap at 100°C for 20 min). 

Leave to cool and filter on a Buchner funnel on glass fiber filter or 
centrifuge. 

Total Attack 

Add the residue of the previous attack (with glass fibre filter) and 2 mL 
of 13 mol (H 2 S0 4 ) L 1 solution in the same 100 mL round bottom flasks 
used earlier. 

Stop the flasks and leave at ambient temperature for 16 h (overnight). 

Carefully add 50 mL inorganic water while cooling. 

Connect the flasks to the condensors and heat at reflux for 5 h 
(alternatively, heat at 90°C in a closed bottle). 

Filter as mentioned earlier on glass wool. 

For the analysis of total sugars, the filtrates described in "Attack 
Favouring the Measurement of Pentoses" earlier and in "Total Attack" 
can be mixed. The analysis of each hydrolysate releases (a) sugars that 
are easily hydrolysable (mainly pentoses) in the hydrolysate described in 
"Attack Favouring the Measurement of Pentoses", (b) sugars like 
cellulose that are difficult to hydrolyse in the hydrolysates described in 
"Total Attack". 

Simplified Total Attack 

In a 100 mL round bottom flask place 5 g of soil sample and 2 mL of 13 
mol (H 2 S0 4 ) L" 1 solution. 

Stop the flask and leave at the ambient temperature for 16 h 
(overnight). 

Carefully add 50 mL inorganic water while cooling. 

Connect the boiling flasks to the condensors and heat at reflux for 5 h 
(alternatively, heat at 90°C in a closed bottle). 

Filter as mentioned earlier on glass wool or centrifuge. 



462 Mineralogical Analysis 

13.2.2 Purification of Acid Hydrolysates 



Principle of the Technique and Main Difficulties 

Particularly for chromatographic titration, the hydrolysates must be 
purified to remove a significant amount of inorganic compounds 
especially sulphate ions but also inorganic dissolved solids (particularly 
iron and aluminium) by the acid attack of the soil. Neutralization of the 
extracts involves the precipitation of aluminium and iron hydroxides. If 
neutralization is carried out with a carbonate or hydroxide belonging to 
the alkaline earths (Ca, Sr or Ba) sulphates can also be precipitated. The 
risk of error lies in the possible adsorption of neutral sugars by the 
precipitates. With barium hydroxide or carbonate, neutral sugars and 
barium salts of uronic acids can be recovered by filtration and by washing 
the precipitates with water, sometimes by washing with hot water or by 
ethanol extraction with a Sohxlet apparatus (Cheshire and Mundie 1966). 
Sodium bicarbonate has also been used for neutralization of the acid 
extracts with methanol extraction of the sodium sulphate after desiccation 
(Oades 1967). 

Calcium carbonate enables neutralization by eliminating sulphates 
together with most coloured organic matter (Brink et al. 1960). Oades et 
al. (1970) preferred strontium carbonate to bring the hydrolysates towards 
pH 7. When the solutions were left to stand, these authors observed co- 
precipitation of brown organic matter and iron complexes. After filtration 
and dry evaporation of the filtrate in a rotary evaporator, sugars were 
dissolved in 4-5 fractions of 2-4 mL methanol with filtration. 

The extracts can be also purified by successive passage on cation and 
anion resin which also separates neutral sugars and uronic acids. 
Alternatively, columns filled with a mixture of coal and celite enable 
sugars to be eluated with 50% ethanol in water after the salts are rinsed in 
water (Cheshire 1979). 

Tests were carried out at IRD 1 (unpublished data) to compare seven 
methods of purification: 

- Water elution on MB 1 amberlite resin, 

- Methanol elution on MB 1 amberlite resin, 

- Neutralization with strontium carbonate, 

- Neutralization with sodium bicarbonate, 

- Neutralization with barium carbonate, 



1 IRD, Institute of Research for Development (ex-Orstom), BP 64501, 911 
Avenue of Agropolis, 34394 Montpellier Cedex 5, France. 



Non-humic Molecules 463 

- Neutralization with barite and 

- Neutralization with soda. 

The method which provided the best recovery of sugars was 
neutralization with strontium carbonate; this result is in agreement with 
thatofOadesetal. (1970). 

Corrective Factors 

Studies using synthetic solutions enabled the effect of neutralization with 
strontium carbonate on the recovery of sugars to be identified (Pansu 
1992). In three tests, the internal standard (myoinositol) was added before 
neutralization, and in three other tests, myoinositol was added before the 
borohydride reduction of the purified concentrated solutions. Each final 
solution was injected twice into the chromatograph. Calculations of the 
absolute contents showed: 

- The error due to the method of preparation of the samples was no 
higher than the error in the chromatographic measurement (F test). For 
most sugars, this error was about 1%. 

- A higher rate of adsorption of the internal standard on the precipitate 
than adsorption of the other sugars. Thus it is better to introduce the 
internal standard after purification of the extracts but before 
derivatization of sugars. 

- An average percentage of recovery of sugars of 85%, ranging from 71% 
for ribose to 91% for rhamnose. Ribose was always recovered in the 
smallest quantities. Based on these recovery percentages, we propose 
the following corrective factors: multiply the results of absolute 
contents of each sugar by 1.21 for rhamnose, 1.08 for fucose, 1.41 for 
ribose, 1.10 for arabinose and xylose, 1.25 for mannose, 1.21 for 
galactose and 1.15 for glucose. These factors take into account the 
response coefficients of each sugar compared to the internal standard. 

Equipment and Reagents 

- Six 100 mL beakers; 

- Magnetic stirrers; 

- Six 250 mL round bottom flasks for the rotary evaporator and 

- Strontium carbonate, M= 147.63 g. 

2.2.4 Procedure 

Transfer the filtrates or centrifugation solutions described in Sect. 13.2.1 
in 100 mL beakers, agitate on a magnetic stirrer. Add the quantity of 



464 Mineralogical Analysis 

strontium carbonate in powder form calculated according to the quantity 
of sulphuric acid to be neutralized plus 10%, i.e.: 

- Hydrolysis A: 1.1 x 40 x 2.5 x 147.63/1000 = 16.2 g 

- Hydrolysis C: 1.1 x2x 13 x 147.63/1000 = 4.2 g 

- Hydrolysis B: the same as hydrolysis c + 2 mL of 2.5 mol L" 1 
acid solution which impregnates the a residue, i.e. a total of 4.9 g. 

SrC0 3 should be added to the solution slowly under agitation to avoid 
foam overflow. 

Leave in contact for at least 1 h under agitation. Check the pH is 
neutral or slightly basic (add a soda pellet if necessary). Check for 
precipitation of iron hydroxides which colour the precipitate and 
discolour the solution. 

Centrifuge and collect the supernatant in a round bottom boiling flask 
for later titration by gas chromatography (GPC) if required (cf. Sect. 
13.2.4), and in a 100 mL volumetric flask if not. Rinse the residue with 
25 mL methanol while stirring with a glass rod. Centrifuge again and add 
the supernatant to the previous solution. Repeat this operation once more. 

13.2.3 Colorimetric Titration of Sugars 



Techniques 

Most colorimetric methods are based on one of the two following 
properties of sugars: their reducing power or, in strong acids, the 
formation of furfural-type compounds that react easily giving coloured 
derivatives. 

The methods can be classified in different categories corresponding to 
the measurement of total sugars, hexoses or pentoses. 

Colorimetry of Total Sugars 

Two titration techniques based on the reducing power of carbohydrates 
were tested (a) reduction of alkaline cupric salt solutions (Fehling's 
liquor) resulting in Cu + cooper complexes or (b) reduction of yellow 
ferricyanide solutions resulting in colourless ferrocyanides. The latter 
was considered preferable for the measurement of soil sugar and was 
automated by Cheshire and Mundie (1966). 

Three techniques use the other property based on furfural derivatives: 
total sugars with anthrone, phenol or orcinol. Anthrone produces a 
beautiful green-blue colour when it comes in contact with the furfural 
derivatives in the concentrated sulphuric acid. This reagent provides 
the best absorbance of complexes with deoxyhexose and hexose 
sugars (except for mannose). However the response to pentose sugars is 



Non-humic Molecules 465 

weaker and becomes undetectable when the anthrone content is above 
0.05%. This method is also suspected of interfering with other organic 
matter as well as with iron and nitrates (Cheshire 1979). 

Phenol reacts with the furfural derivatives and results in a yellow 
colouration (Dubois et al. 1956). The similarity of this colour to that of 
soil hydro lysates could result in overestimation of total sugars with this 
reagent (McGrath 1973). Overestimated data were observed with direct 
phenol colorimetric analysis without purification of hydrolysates (Pansu 
1992). However, Doutre et al. (1978) considered this method more 
satisfactory than the anthrone method. 

Orcinol or 3,5-dihydroxytoluene also reacts with the furfural 
derivatives, in this case with the advantage of providing enough similar 
responses for each sugar. It was used for soil hydrolysates by Bachelier 
(1966). 

Colorimetry of Hexose Sugars 

Although originally proposed for total sugars, anthrone is more 
commonly used to measure hexoses and deoxyhexoses of soil hydro- 
hydroly sates. 

Ivarson and Sowden (1962) also proposed chromotropic acid for the 
measurement of hexoses. This reagent is not very susceptible to 
interference with pentose sugars and uronic acids and responded more 
strongly than anthrone on four samples of soil and litters. 

Colorimetry of Pentose Sugars 

Cheshire and Mundie (1966) used the orcinol-FeCl3 reagent described by 
Thomas and Lynch (1961) to measure the maximal release of pentose 
sugars during hydrolysis. In acetic acid, aniline also reacts with pentose 
sugars at ambient temperature resulting in a red colouration, with only 
slight interference by hexose sugars and uronic acids (Ivarson and 
Sowden 1962; Tracey 1950). 

Colorimetry of Deoxyhexose Sugars 

The yellow colour formed by heating the carbohydrate extract with 
cysteine in sulphuric acid medium is quoted as specific to deoxyhexose 
sugars; this reagent was used for soil hydrolysates by Cheshire and 
Mundie (1966). 

See Chap. 14 for titration of uronic acids and amino sugars. 

Equipment and Reagents 

- Calibrated 150^25 mm glass test tubes; 

- Water bath; 



466 Mineralogical Analysis 

- Visible spectrophotometer; 

- Plastic colorimetric cells, length 1 cm; 

- Crushed ice; 

- Concentrated sulphuric acid 18 mol (H 2 S0 4 ) L" 1 (d= 1.84); 

- 5% phenol solution in water; 

- 0.2% solution of anthrone in concentrated H 2 S0 4 ; 

- Standard solutions for the phenol method: 0, 5, 10, 25, 50 and 100 mg 
(glucose) L" 1 and 

- Standard solutions for the anthrone method: 0, 5, 10, 15, 20 and 25 mg 
(glucose) L 1 . 

Procedure for the Phenol Method 

Put in the test tubes 2 mL of soil hydrolysate (cf Sect. 13.2.1) or purified 
soil hydrolysate (cf. Sect. 13.2.2) that has been previously completed to 
100 mL in a volumetric flask (a preliminary test of standard additions can 
be used to check the need for purification). 

Add 1 mL phenol solution and then rapidly add 5 mL of concentrated 
sulphuric acid without allowing it to run along the wall of the flask 
(taking care not to splash). 

Leave to stand for 10 min, agitate the tubes and place them in the water 
bath at 25-3 0°C for 20 min, then cool under running water. 

Read absorbance at 485 nm (490 nm for hexose sugars, 480 nm for 
pentose sugars and uronic acids). The colour remains stable for several 
hours. It is sometimes necessary to homogenize the solutions just before 
colorimetric reading. 

Proceed in the same way for each point of the calibration range. 

Procedure for the anthrone method 

Bring the hydrolyzed solution (cf. Sects. 13.2.1 or 13.2.2) to 100 mL and 
homogenize well. 

Introduce 5 mL of this solution in a calibrated test tube placed in ice. 
Proceed in the same way for each calibration point. 

Slowly add in each tube 10 mL of anthrone solution letting it run down 
the side of the tube; swirl the tube to mix. 

Seal with a piece of parafllm and immediately place in a 85°C water 
bath for 35 min. 

Cool in an ice-tray and place in a colorimetric cell and read absorbance 
at at 625 nm. Disposable plastic colorimetric cells (1 cm in length) 
should be used to limit the number of transfers and washings of the 
concentrated sulphuric acid solutions. 



Non-humic Molecules 467 

13.2.4 Titration of Sugars by Gas Chromatography 



Principle 

The titration of sugars by gas chromatography is rather difficult to 
implement for two reasons: 

- Sugars are too polar to be satisfactorily separated directly on the gas 
chromatographic columns; it is thus necessary to form derivatives 
which transform the hydroxyl functional groups into less polar forms; 

- Gas chromatography is more selective than liquid chromatography; 
separation of the carbohydrate can result in many peaks representing 
the isomers of the different molecular configurations and the 
chromatograms may then be difficult to interpret. 

Most authors used techniques similar to the one described by Oades 
et al. (1970). Sodium borohydride was added to the neutralized and purified 
acid extracts, and this transformed the isomers of sugars into their alditol 
form. After elimination of the boric acid formed by successive 
evaporations in acetic acid medium, acetylation of the alditols was 
performed with acetic anhydride. 

Alditol acetates were dissolved in methylene chloride for injection into 
the chromatograph. Oades et al. (1970) used a 2 m column with an 
interior diameter of 3.5 mm filled with 100-120 mesh GAS Chrom Q 
impregnated with 5% ECNSS-M. In this way eight major soil sugars 
were separated in 70 min though with rather poor distinction between 
rhamnose and fucose. 

Spiteller (1980) improved this technique. He separated alditol acetates 
on a non-polar OV1 25 m capillary column in 25 min by detecting traces 
of glucosamine and galactosamine. Cheshire et al. (1983) separated 
alditol acetates with a capillary column of 50 m x 0.3 mm 
impregnated with SILAR 10 CP with a 90 min temperature 
programme. Dormaar (1984) obtained separation with a duration similar 
to that used by Spiteller (1980) with a glass capillary tube impregnated 
with SP2330. Baldock et al. (1987) used the procedure of Spiteller, as did 
Arshad and Schnitzer (1987), the latter authors with a capillary tube and a 
stationary phase with trifluoropropyl-methyl which increased the total 
duration of the analysis. Coelho et al. (1988) used a filled column, and 
the length of the separation phase was similar to that of Oades et al. 
(1970). Like Cheshire and Griffiths (1989) and Murayama (1988) used 
chromatographic separation of alditol acetates but did not specify the 
column used. 

The reduction and acetylation operations which follow the purification 
of the hydrolysates are rather long and this means many samples cannot 



468 Mineralogical Analysis 

be compared. Blakeney et al. (1983) recommended a method allowing the 
preparation time to be reduced, but a test performed at the IRD laboratory 
with this aim in view was unsuccessful (unpublished data). 

On the other hand, the chromatographic time (Fig. 13.1) was reduced 
to 12.5 min using a capillary column impregnated with a SP2330 phase 
similar to that of Dormaar (1984), but made of silica glass instead of 
borosilicate glass (Pansu 1992). 

Equipment and Reagents 

- 5 mL conical cylinder flasks with PTFE joint screw caps (Fig. 13.2); 

- Pasteur pipettes with 3 mL squeeze bulbs; 

- Thermostated aluminium heating block (Fig. 13.2); 

- Nitrogen sweeping for evaporation (Fig. 13.2); 

- Gas phase chromatograph equipped with a flame ionization detector; 

- SP2330 silica capillary column (Supelco) 15 m in length and 0.25 mm 
ID; 

- Standard carbohydrate solution in 85% methyl alcohol containing 1 mg 
mL -1 of each carbohydrate: rhamnose, fucose, ribose, arabinose, xylose, 
mannose, galactose and glucose; 

- Standard solution containing 1 mg mL" 1 myoinositol in 50% methyl 
alcohol; 

- Methyl alcohol; 

- Strontium carbonate; 

- Sodium borohydride; 

- Glacial acetic acid; 

- Anhydrous solution of 10% acetic acid in methanol (dry on anhydrous 
Na 2 S0 4 ); 

- Acetic anhydride and 

- Chloroform. 

Preparation of Alditol Acetates 

(a) In each boiling flask containing the purified hydrolysates, add an 
exact volume of the myoinositol internal standard solution appropriate 
for the estimated quantity of sugars (0.5-2 mL). 

(b) Evaporate to just dry with a rotary evaporator; rinse the boiling flask 
with 3-4 small fractions of methanol using a Pasteur pipette and 
transfer the washing solutions to a 5 mL flask with screw cap. 

(c) Add approximately 10 mg of sodium borohydride and leave to act 
overnight. 

(d) Add 0.1 mL of glacial acetic acid, evaporate to dry at 70°C under a 
nitrogen flow (Fig. 13.2), add 1 mL of anhydrous 10% acetic acid in 



Non-humic Molecules 469 

the ethyl alcohol solution and evaporate in the same way, repeat this 
operation five times. 

(e) Add 1 mL acetic anhydride, stop the flasks and heat at 135°C for 2 h. 

(f) Cool to 70°C and evaporate to dry under a nitrogen flow. 

(g) Cool and dissolve in an exact volume of from 0.5 to 2 mL of 
chloroform depending on the estimated sugar content. 

The solutions can be stored or injected directly into the chromatograph. 

Preparation of Standards for Aid itol Acetates 

Add 1 mL standard solution of sugars and 1 mL of internal standard 
solution in a 5 mL flask with a screw cap and continue as mentioned 
earlier starting at stage c. 

Chromatographic Conditions 

- Silica glass Supelco SP2330 capillary column (or similar) length: 15 m, 
interior diameter: 0.25 mm. 

- Carrier gas: 0.7 bar helium. 

- Splitter injector, leak-flow: 100 mL min 1 . 

- Injection: 1 jlxL. 

- Flame ionization detector. 

- Temperatures: 

column programmed from 210 to 250°C at 3°C min" 1 , 
injector: 300°C, 
detector: 250°C. 

Figure 13.1 shows an example of chromatograms obtained on sugars in 
a standard solution and in a ferrallitic soil from Congo. 



470 



Mineralogical Analysis 



FU AR GA 

RH Rl XY MA GL MY 



NJ 



u 



V 



GAMY 
AR XY MA GL 



1 I 


1 


i 


i 1 i 





4 


8 


12 


210 
I i 


222 

l 


234 
1 


246 
I i 





210 



222 

I i 



8 
234 



12 Min 

246 °C 

_J i 



Fig. 13.1. Chromatograms of alditol acetates (conditions described in "Chromato- 
graphic conditions", RH, rhamnose; FU, fucose; Rl, ribose; AR, 
arabinose; XY, xylose; MA, mannose; GA, galactose; GL, glucose; 
MY, myoinositol internal standard). On the left: standard mixture 
corresponding to the injection of 0.2 jig of each sugar. On the right: 
sugars of a strongly desaturated ferrallitic soil of the Niari valley 
(Congo). Hydrolysis according to the procedure described in "Attack 
Favouring the Measurement of Pentoses" earlier. 



Non-humic Molecules 



471 



Platinum 

temperature 

sensor 




- Screw cap 
PTFE joint 

5mL graduated tube 



o o o o o 



Regulation 
system 



C-On On 



C-Off Off 



Aluminium heating 
block 
Reacting 
compounds 

Regulated 
/ heating plate 




Resistive heater 
Cooling system 



Solenoid valve 
Water 



Inox tube 
1/16' 





o o o o o 



C-On On 
C-Off Off 



© 



CL 



Distribution 

tube 

1/4" 




Fig. 13.2 Recommended system for derivatization reactions for GPC. 
Top, reactions in closed micro-flasks at controlled temperature, 
bottom, evaporation of excess solvents and reagents under surface 
nitrogen flow 



472 Mineralogical Analysis 

13.2.5 Quantification of Total Lipids 

Principle 

Total lipids are measured gravimetrically after extraction with organic 
solvents in a Soxhlet extractor. Most lipidic compounds of soils are not 
very polar, but some are slightly more so (Ambles et al. 1990). The 
choice of the polarity of the extraction solvent is thus significant, and 
preliminary tests should be performed. Fahd-Rachid (1993) used 
chloroform. A mixture of petroleum ether and ethyl acetate (3:1, v:v) is 
also often recommended and its toxicity is lower than chlorinated 
solvents. However, this mixture was considered to be less effective than 
chloroform for the extraction of complex lipids (Jambu et al. 1987). 

Solvents do not extract all the lipids since some are associated with 
humic compounds, clays and various minerals. An acid treatment enables 
release of the bound lipids which then become accessible for a second 
Soxhlet extraction. Hydrochloric acid can be used for the acid pre- 
treatment but according to a technique recommended by Wang et al. 
(1969), it is better to use a mixture of hydrochloric acid and hydrofluoric 
acid. Hydrochloric acid enables release of lipids bound to cations, and 
hydrofluoric acid enables release of lipids bound to the organomineral 
matrix (Fahd-Rachid 1993). 

Equipment and Reagents 

- 200 mL Soxhlet extractor with condenser and a 500 mL borosilicate 
glass round bottom boiling flask with conical ground joints; 

- Hemispherical electric heating mantle with temperature regulation for 
use with a 500 mL boiling flask; 

- Soxhlet extraction cartridges 38 mm in diameter and 150 mm in height; 

- Vacuum rotary evaporator; 

- Pasteur pipettes; 

-25 mL half cylindrical half conical flasks of the volumetric type 
(graduation is not necessary) with PTFE joint screw cap; 

- Glass funnels (/> 3 cm; 

- 250 mL plastic beakers; 

- Plastic funnels <j> approx. 8 cm; 

- Filter papers for plastic funnels; 

- HCL-HF aqueous solution at 2.5% HF and 2.5% HC1 and 

- Petroleum ethenethyl acetate extraction solution (3:1 v:v): mix 750 mL 
petroleum ether and 250 mL ethyl acetate. 



Non-humic Molecules 473 

Procedure 

Free Lipids 

- Put an exact weight P (100-150 g) of air-dried soil sieved to 2 mm in 
the extraction cartridge and put the cartridge in the Soxhlet extractor. 

- Put 200 mL extraction solution and two pumice grains in the 500 mL 
boiling flask; connect the boiling flask to the Sohxlet extractor. 

- Connect the condensor to the top of the extractor. 

- Start heating the boiling flask and regulate the heat of the hemispherical 
mantle so as to obtain good condensation of the solvent from the 
condenser to the extractor. 

- Continue the extraction for 48 h. 

-Let cool then connect the boiling flask to the rotary evaporator and 
evaporate until the volume is reduced to a few mL. 

- At the same time, tare a stopped 25 mL half cylindrical half conical 
flask (after drying in the drying oven and cooling). 

- Open the flask and position it under a small funnel stopped with a glass 
wool plug and half filled with anhydrous sodium sulphate. 

- Using a Pasteur pipette, decant the residue of evaporation into the small 
funnel and collect the dried extract in the 25 mL flask. 

-Rinse the evaporation flask and the funnel several times with a few 
Pasteur pipettes of the extraction mixture to fill approximately 20 mL 
of the 25 mL flask. 

- Evaporate the solvent from the flask under gaseous nitrogen flow (see 
Fig. 13.2) or under vacuum in the rotary evaporator by means of a 
ground/screwed connection. 

- Stop the flask, let cool and weigh, by deduction of the tare one obtains 
the weight of free lipids P\. 

- Rate of free lipid = 100 P X IP. 

Bound Lipids 

- Place the residue of extraction of the free lipids in a 250 mL plastic 
beaker and add the HCL-HF solution at a rate of 150 mL for a test 
sample of 100 g soil. 

- Leave in contact for 48 h agitating from time to time. 

- Filter on filter paper in plastic funnels. 

- Wash the residue with water until pH 5; decant it to a shallow cup or 
glass beaker cover and leave to dry on the lab table or in a desiccator. 

- Extract the lipids using same procedure as for free lipids (cf "Free 
Lipids" earlier). 



474 Mineralogical Analysis 

Total Lipids 

It is possible to extract the total lipids directly by an attack of the initial 
sample with the HCL-HF mixture followed by extraction in the Sohxlet. 
Fahd-Rachid (1993) found excellent agreement between the total 
extracted lipids and those calculated by the sum "free lipids + bound 
lipids". 

13.2.6 Quantification of the Water-Soluble Organics 

Table 13.1.1. Cold water soluble (CWS) and hot water soluble (HWS) compounds 
obtained on cultivated soils of Boigneville, France (Pansu, 
unpublished data, see extraction conditions in the text) 

M T g ? C o/o in C r W M S CWS- weight % 9* H p W M S % ™ S ", 

Na %°f CWS C i N C/total of HWS '" C i N C/total so.l 

CWS ratio .. ~ HWS ratio C 

soil C 



A 


0.026 


28.8 


2.1 


0.75 


0.85 


8.3 




7.1 


B 


0.033 


33.8 


2.5 


1.12 


0.48 


22.3 




10.7 


D 


0.004 


75.0 


2.1 


0.30 


0.26 


21.5 




5.6 


E 


0.021 


27.1 


1.4 


0.57 


0.24 


25.1 


5.5 


6.0 


F 


0.025 


30.1 


1.8 


0.75 


0.38 


22.4 




8.5 



For the study of the spatial and temporal dynamics of organic matter, 
the water-soluble organic fractions of the soil have to be taken into 
account. These fractions comprise organic acids, simple sugars or light 
polysaccharides and nitrogenous compounds. These different compounds 
can have a significant influence on the structural stability and fertility of 
the soils. Two types of compounds can be distinguished: 

- Compounds that are soluble in cold water: these are obtained by 
agitating the soil with water using different procedures, for example, 
shake 10 g of soil in 200 mL water or 2 h on a rotary shaker, leave in 
contact overnight, sake again for 2 h and centrifuge at 14,000 g. 

- Compounds that are soluble in hot water: several procedures can be 
used, for example, boil 4 g of soil at reflux for 16 h with 200 mL water 
in the presence of three glass balls, cool and centrifuge at 14,000 g. 
Kouakoua et al. (1997) showed that the extraction of water-soluble 
compounds of ferrallitic soils from Congo increased continuously with 
the length of extraction both in a drying oven and in an autoclave. 
According to Leinweber et al. (1995), this fraction is mainly made up 
of nitrogenous and carbohydrate compounds. 

Table 13.1 lists proportions of these fractions expressed in mass, 
carbon and nitrogen contents for five cultivated soils in temperate zones. 



Non-humic Molecules 475 

In waterlogged or poorly aerated soils, the aqueous extracts contain 
organic acids of low molecular weight (e.g. lactic, pyruvic or acetic acid) 
under anaerobiosis conditions (Kiisel and Drake 1999) which can be 
titrated in aqueous mediums by gas-solid chromatography (standard 
column of Porapak or chromosorb 101, or similar) or by ionic chromato- 
graphy. The aqueous extracts contains also inorganic water soluble 
compounds (cf. Chap. 18). 



13.3 Complementary Techniques 

13.3.1 Determination of soil Carbohydrates by Gas 
Chromatography 

If the alditol acetate technique (cf. Sect. 13.2.4) was the most widely used 
for the measurement of soil carbohydrates, other techniques have also 
been proposed: Morgenlie (1975) described separation of neutral sugars 
in the form of their O-isopropylidene derivatives. Preparation of the 
derivatives was performed with 1% sulphuric acid in acetone reagent, and 
separation required 35^10 min on columns of the XE60 or OV225 type. 
Traitler et al. (1984) separated trimethylsilyl derivatives of sugars on 
short apolar columns. Cowie and Hedges (1984) also separated trimethyl- 
silyl derivatives from sugars from hydrolysates in plankton, sediment and 
wood. As a preliminary treatment, these authors brought free sugars to 
their mutarotation equilibrium in the presence of lithium perchlorate to 
get round the problem of multiple peaks. Larre-Larrouy and Feller (1997) 
and Larre-Larrouy et al. (2003) analyzed neutral sugars in soil at the 
same time as uronic acids and hexosamines in the form of their tri- 
methylsilyl derivatives, each sugar being calculated by the sum of the 
surfaces of its different isomer forms. 



13.3.2 Carbohydrates by Liquid Chromatography 

The techniques used for the titration of sugars with liquid chromato- 
graphy were unsatisfactory for many years. Cheshire et al. (1969) 
separated eight neutral sugars by ion exchange chromatography with a 
pH gradient, but separation took 14 h. At the column exit, sugars were 
analyzed by colorimetry after reaction with the orcinol in the sulphuric 
acid reagent. Hydrazide of /?-hydroxybenzoic acid is a better reagent for 
alkaline eluates; without acidification it gives an yellow colour with 
carbohydrates. 



476 Mineralogical Analysis 

Hamada and Ono (1984) analyzed sugars on soils of volcanic ash by 
high performance liquid chromatography (HPLC) with an anion column 
and detection by fluorescence spectroscopy after reaction with 
ethanolamine. Separation required 70 min but arabinose, fructose and 
fucose were eluted under the same peak, as were rhamnose and ribose. 
Pluijmen (1987) analyzed sugars of different plants by HPLC on a 
SUGAR PAK TM column (Waters Associates) with a water (or 
acetonitrile-water) mobile phase, refractometric detection and an anion 
and cation precolumn. But this author was especially concerned with 
glucose and fructose and did not provide chromatograms. 

Reim and Van Effen (1986) used a technique that appeared to be more 
promising. They proposed a new detector that was more sensitive than 
the refractometer and which, in addition, did not require preliminary 
derivatization reactions as do spectrometric methods. Using an ion 
exchange column, they simultaneously separated simple sugars and low 
molecular weight oligomers, but they did not provide a chromatogram of 
the eight main soil sugars. 

Angers et al. (1988) separated sugars from hydrolysates of soils on an 
aminex HPX-87P column (BIO-RAD labs); but they titrated only five 
sugars and the detection limit was rather poor. 

Martens and Frankenberger (1990) separated ten soil sugars by anion 
exchange chromatography using the HP AC-PAD system (Dionex, 
Sunnyvale, CA, USA) with the following chromatographic conditions 
(Fig.13.3): 

- 200 |LlL injection loops; 

- CarboPac PA guard column (25 x 3 mm); 

- Chromatographic column (250 x 4 mm) filled with a pellicular anion 
exchange resin: CarboPac PA1; 

- Flow of eluent: 0.8 mL min " , ambient temperature; 
eluent a: purified inorganic water (18 Mohm), 

eluent b: 50 mmol (NaOH) L" 1 + 1.5 mmol (CH 3 COONa) L" 1 aqueous 

solution, 

93% eluent a and 7% eluent b for 15 min, gradient up to 100% b in 25 

min, 

idem the mobile phase has to be degassed to prevent absorption of C0 2 

and the production of carbonates which can move ions and reduce the 

retention time; 

- Detection by pulsed amperometry three times with a gold electrode: 
£1:0.1 V, t\\ 300 ms, oxidation of CHOH groups, 

E2: 0.6 V, tl\ 120 ms, displacement of the reaction products, 
is3:-0.8 V, t3: 300 ms, cleaning of the electrode at negative potential, 
response time: 1. 



Non-humic Molecules 



477 



The study of Martens and Frankenberger (1990) showed that the 
HP AC-PAD system had several advantages over another classical HPLC 
system with detection by refractive index: it produced more precise 
results, was twice as sensitive (pmole) and had better resolution. The 
preparation of the soil samples was also simpler in the HP AC-PAD 
system. After acid attack (cf. Sect. 13.2.1), the samples were treated with 
1 mL 0.1 mol (EDTA) L" 1 solution then brought to pH 4 by adding 5 
mol (NaOH) L" 1 solution and centrifuged at 10,000 g. The coloured 
materials were then removed by filtration on a solid phase extraction 



column (SPE) (Supelco Bellefonte, Pa, USA) comprising 3 mL of 
strong cation exchange resin (three propylsulfonic acid, H + form) and 
3 mL of strong anion exchange resin (quaternary propylammonium 3, Cl- 
form). The extracts were also filtered on 0.22 |Lim GS filters (Millipore, 
Bedford, MA, USA). 




10 15 20 

Retention time (min) 



25 



30 



Fig. 13.3. Standard sugar separation by high performance anion exchange 
chromatography (Martens and Frankenberger 1990); Ino, inositol; 
Rib, ribitol; Fu, fucose; Rh, rhamnose; Ga, galactose; Gl, glucose; 
Xy, xylose; Ma, mannose; Ri, ribose; Lac, lactose 



Bernal et al. (1996) proposed a chromatographic technique similar to 
that of Martens and Frankenberger (1990) for sugar titration in coffee and 
wine. 



478 Mineralogical Analysis 

13.3.3 Fractionation and Study of the Soil Lipid Fraction 

Lipid Fractionation 

There are many lipid fractionation techniques and the reader is advised to 
consult relevant handbooks that describe biochemical lipidology 
techniques, e.g. Kates (1975). 

Fractionation can be divided into two stages: 

- Separation of the different classes of lipids and 

- Measurement of the individual components of each class or of the total 
fraction. 

The exact procedure to apply depends on the lipid concerned. Most 
lipids of microbial and animal origin contain from 60 to 85% of 
phosphatides and glycolipides, the remainder being neutral or not polar 
lipids (glycerides, sterols, hydrocarbons, pigments). Lipids of plant origin 
contain a larger proportion of neutral lipids and a smaller proportion of 
phosphatides. 

Two main types of techniques are used to separate the classes of lipids: 

- Fractionation by solvents and 

- Liquid phase chromatography on column, on paper or on thin layer. 

The characterization and quantification of lipid components require the 
chemical breakdown of complex lipids followed by separation techniques such 
as gas chromatography, generally with a flame ionization detector, possibly 
coupled with a mass spectrometer for the identification of the molecules. 

Fractionation by Solvents 

Precipitation by acetone is the simplest method for separating phosphatides 
and neutral lipids. It is based on the insolubility in cold acetone (0°C) of 
most phosphatides (particularly of acid phosphatides in salt forms), whereas 
neutral lipids are soluble. This procedure is well suited for lipids of animal 
and microbial origin, but is less effective in the presence of high proportions 
of neutral lipids like most lipids of plant origin (Kates 1975). 

The techniques for separation of nonmiscible pairs of solvents (polar 
and non-polar) are also useful, especially for lipids rich in glycerides. As 
the first stage of the soil lipid fractionation, Stevenson (1982) recom- 
mended the separation of chloroform and an aqueous soda solution. The 
aqueous phase was then acidified and extracted with ether to recover the 
free fatty acids. The chloroform phase contains the other lipids which 
were then separated by chromatography. 

Fractionations by Liquid Phase Chromatography 

Many methods have been proposed. For preliminary fraction-ation of the 
total lipidic extract of the soil, Jambu et al. (1991, 1993) and Ambles 
etal. (1989, 1990) used the technique recommended by McCarthy and 



Non-humic Molecules 479 

Duthie (1962). Lipids were separated on columns of potassic silica 
(silicic acid treated with potash in isopropanol) in three fractions: neutral 
(the first elution with ethyl ether), acid (elution with 2% formic acid in 
ether solution) and polar. Zelles and Bai (1993) used a similar technique 
with SPE. 

It was then possible to separate the neutral fraction of lipids on silica 
columns (Kiesselgel 60, Merck, Jambu et al. 1993) or Florisil treated or 
not with acid (Kates 1975). Elution was carried out initially by hexane or 
petroleum ether to separate hydrocarbons then by mixtures of increasing 
polarity to split the other classes of lipids. Mixtures with an increasing 
proportion of ethyl ether in petroleum ether were used most frequently 
enabling successive separation of esters (sterilic, methyl esters), ketones, 
triglycerides, diglycerides, monoglycerides, free alcohols and sterols. 

Gas-Liquid Chromatography Techniques 

These techniques are often used for fractionation, characterization and 
quantification of lipid components. They can be applied directly to 
certain fractions like hydrocarbons or after breakdown of the heavy 
lipidic compounds into fatty acids and unsaponifiable products (cf. 
"Fractionation of Fatty Acids and Unsaponifiables"). 

Fractionation of Fatty Acids and Unsaponifiables 

Principle 

A saponification reaction enables breakdown of heavy lipidic substances 
to obtain fatty acids, and other saponification products (like glycerol and 
sterols) with unsaponifiable compounds. The fatty acids are then 
methylated, separated, and quantified by Gas-Liquid Chromatography 
(GLC), the unsaponifiables are also titrated by GLC after silylation of the 
hydroxyl groups. The technique can be used for total lipidic extracts or 
for lipid groups that have already been separated as described in "Lipid 
Fractination" earlier. 

Equipment and Reagents 

-Fl flasks: 25 mL half cylindrical half conical borosilicate glass flasks, 
(volumetric flasks without graduation, cf "Equipment and Reagents" 
earlier under Sect. 13.2.5 and Fig. 13.4) with PTFE screw cap. 

- F2 flasks: 25 mL half cylindrical half conical flasks with PTFE screw 
cap. 

-F3 flasks: 10 mL half cylindrical half conical borosilicate glass flasks, 
(volumetric flask without graduation) with PTFE screw cap. 



480 Mineralogical Analysis 

- F4: half cylindrical half conical borosilicate glass volumetric tubes with 
PTFE screw caps. 

- Funnels with a diameter of 3 cm. 

- Pasteur pipettes. 

- 0.1 mg precision balance. 

- Gas phase chromatograph with splitter or "split- splitless" injector for 
capillary column and detection by flame ionization detector. 

- Anhydrous petroleum ether. 

- Anhydrous methanol (stored on anhydrous sodium sulphate). 

- 0.3 mol (NaOH) L" 1 solution in methanol: dissolve 1.2 g of soda in 100 
mL anhydrous methanol. 

- 3% H 2 S0 4 methanolic solution: add 3 mL of concentrated sulphuric 
acid in a 100 mL volumetric flask containing 70 mL anhydrous 
methanol, agitate and cool, adjust to 100 mL and stop well. 

- 60% methanol in water. 

- Anhydrous sodium sulphate. 

Procedure 

Saponification 

-Add 4 mL of petroleum ether and 2 mL methanol in the 25 mL Fl 

flasks containing the free or bound lipidic extracts (cf "Procedure" 

under Sect. 13.2.5), agitate for 1 h. 

- Add 8 mL of 0.3 mol (NaOH) L" 1 solution in methanol. 

- Stop the flasks hermetically and heat at 100°C for 2h30 in a thermo- 
stated aluminium heating block (Fig. 13.2). 

- Cool, open the flasks and add 60% methanol in such a way that the 
petroleum ether phase is in the upper cylindrical part of the flask. 

- Using a Pasteur pipette, take the upper phase and decant it in a tared 25 
mL conical F2 flask through a small funnel stopped with a glass wool 
plug and filled with a spatula of anhydrous sodium sulphate (Fig. 13.4). 

- Add 3 mL of petroleum ether to the first Fl flask; stop and agitate well 
then remove the upper phase in the same way and add it to the previous 
phase through the same device; repeat this procedure four times. This 
phase contains unsaponifiables and saponification products other than 
fatty acids (F2). 

- Acidify the first flask with 1 mL of concentrated hydrochloric acid 
diluted two times, then extract again with petroleum ether as previously 
described. Decant and collect the upper phase in a tared 10 mL F3 
flask; this phase contains the fatty acids. 



Non-humic Molecules 



481 



25 mL screw 
screw flask: F1 



Water-alcohol 
polar phase 




Squeeze 

bulb 

3ml_ 



Pasteur 
pipette 



Non-polar 
phase 




Anhydrous 
sodium sulphate 

Glass wool 
plug 

Removable 
funnel rack 



Aluminium 

block (Cf. Fig. 13.1) 

25 mL screw 
flask: F2 



Fig. 13.4. Separation by decantation (on the left) and drying of the recovered 
products (on the right) 

Non Fatty Acid Compounds 

- Evaporate the contents of the F2 flask to dry under nitrogen flow or 
with a rotary evaporator. 

- Stop and weigh the flask to obtain the total quantity. 

-Add 100 |LlL of trimethylchlorosilane (TMCS), 300 |LlL of hexamethyl- 
disilane (HMDS) and 600 juL pyridine, stop and heat at 70°C for 5 h 
(Fig. 13.2). 

-Inject 2 jlxL in the gas phase chromato graph under the following 
conditions: 

(a) CPWAX capillary column (or similar) length: 25 m, interior 
diameter: 0.3 mm, temperature 230°C, 

(b) carrier gas He 0.5 B, 

(c) split injector, leak flow 50 mL min" 1 . 



482 



Mineralogical Analysis 



Fatty Acids 

-Bring the contents of the 10 mL F3 flasks to dry and weigh to obtain 

total fatty acids. 
-Add 3 mL of anhydrous 3% sulphuric acid in the methanol reagent; stop 

the flasks and heat at 70°C for 5 h (Fig. 13.2). 

- Let cool and add 2 mL petroleum ether and approximately 3 mL water 
to recover the ether phase in the cylindrical part of the flask (Fig. 13.4). 

- Recover the fatty acid methyl esters in 10 mL F4 tubes using the same 
procedure as for the non-fatty acid lipidic fraction, complete to 10 mL 
and inject 2 jlxL in GPC under the following conditions (short acids < 
C20,Fig. 13.5): 

(a) 25 m x 0.3 mm silica glass capillary column impregnated with 
CPWAX phase (or similar), 

(b) He carrier gas, input pressure 0.6 B, 

(c) Split injector 180°C, leak-flow 60 mL min 1 , 



(d) Temperature programme 180°C - 

(e) Flame ionisation detector, 240°C. 



2°C min 



-> 240 °C, 




Fig. 13.5. Fractionation of methyl esters of short fatty acids of a tropical ferruginous 
soil from Burkina-Faso by gas chromatography (conditions described in 
the text; M Pansu, unpublished data) 



Non-humic Molecules 483 

Notes 

- Some reagents are much faster than MeOH-H 2 S0 4 making it possible 
to obtain fatty acid methyl esters instantaneously at room temperature 
(e.g. BF3-CH3OH or diazomethane); the reagent described in "Procedure" 
under Sect. 13.3.3 earlier was used because it is not expensive and the 
heating system is suitable for derivatization. 

- For soils poor in fatty acids, the 10 mL volumetric half cylindrical half 
conical F4 tubes are very useful for the concentration of the mixture by 
solvent evaporation under nitrogen flow. 

-The chromatographic column used to obtain separation of Fig. 13.5 is 
not suitable for the fractionation of long chain fatty acids of soil (C20- 
C34); for this purpose a shorter column or a different impregnation 
phase is required. 

- The procedure described in "Equipments and Products" later can be 
simplified for the determination of fatty acids alone; use direct 
transmethylation on the lipidic fraction with the H 2 S0 4 -methanol 
reagent; extract the fatty acid methyl esters by decantation in petroleum 
ether after adding water (Fig. 13.4); however, this technique may 
complicate the reading of the chromatograms due to a risk of peaks of 
fatty acid methyl esters overlapping peaks of unsaponifiable compounds. 

13.3.4 Measurement of Pesticide Residues and Pollutants 

Principle 

Like lipids, pesticides and pollutants are soluble in organic solvents. They 
can be extracted using similar techniques (cf Sect. 13.2.5), although 
extraction with the Soxhlet apparatus is not recommended for pesticides. 
Indeed, the molecules are often degradable and prolonged boiling of the 
mixture of solvent and extracted products could lead to underestimation 
of the contents of residues. 

Due to problems of degradability, the most commonly used methods 
use cold extraction in solvents, specially micro-methods (Steinwandter 
1992) and methods using supercritical fluids (Richter 1992; Table 13.2). 
If the soil samples have to be stored before extraction, they should be 
frozen or freeze-dried. 

After the extraction phase, if lipid content is high, purification may be 
necessary. Two techniques are available (a) separation using solvents of 
different polarities and (b) liquid chromatography on a Florisil or alumina 
column. 

It is usually necessary to concentrate the soil extracts as much as possible 
to improve the limit of detection. It is useful to carry out the final 



484 



Mineralogical Analysis 



concentration phase using a gaseous nitrogen flow on the surface of the 
extract in half cylindrical half conical volumetric tubes (cf. Sect. 13.3.3). 

Table 13.2. Some supercritical fluid techniques used for the extraction of soil 
pesticide residues (Richter 1992) 



pesticide 



extraction conditions 



permethrin, atrazine, deltamethrin, 
dieldrin, carbofuran, diuron, 2,4 D, 
methylparathion 



Methanol 250 °C, 150 bars, 1 mL min" 
2h 



DDT 



DDT 



C0 2 , 40 °C, 100 bars, 0.7 g s , 10 min 

C0 2 + 5% methanol or toluene, 40 or 100°C, 0.7 

g s _1 , 5 min 



lindane, aldrin, DDT 



C0 2 , 138 bars, 15 min 



linuron, diuron 



C0 2 + methanol, ethanol or acetonitrile, 75 to 
120°C, 100-400 bars, 2.5-8.5 mL min" 1 , 15-180 
min 



sulfonylurea 



C0 2 + 2% methanol, 40°C, 223 bars, 
6 mL min" 1 , 2-15 min 



simazme, atrazine, propazme, 
terbutylazin, cyanazine 



C0 2 to 48°C, 230 bars, 1.7 mL min" 1 , 
30 min 



organochlorides, organophosphorous 



C0 2 100% and C0 2 + 10% methanol, 50-70°C, 
150 or 300 bars, 25-60 min 



DDT, DDE, DDD, lindane, aldrin 



C0 2 + 5% acetone, 75°C, 400 bars, 60 min 



The recommended choice for the analysis of the extract is gas 
chromatography because of (a) its good selectivity, particularly with 
capillary columns and (b) the large number of selective and very sensitive 
detectors that are available. The recommended injector for most non- 
volatile pesticides is a glass-needle injector which ensures the maximum 
possible concentration of the solutes during injection. Alternately split- 
splitless injector can be used. Other techniques are based on HPLC, 
generally with UV detection. 

The diversity of the pesticides and pollutants present in the soil (cf. 
Sect. 13.1.4 later) has resulted in the development of a very large number 
of specific analytical procedures which cannot be exhaustively described 
here. Based on the literature and laboratory practice, a choice of 
procedures for the extraction, purification and chromatographic analysis 
of the main families of products is provided later. In the future, these 
procedures will change as a result of further advances in analytical 
chemistry and some of the recommendations later will need to be 



Non-humic Molecules 485 

updated. For example, Gennaro et al. (1996) presented the simultaneous 
separation of phenoxyacetic acid, triazins and phenylureas herbicide 
residues in soils by HPLC. Celi et al. (1993) proposed a way of 
measuring fenoxatrop and ethyl fenoxatrop in a range of soils. Anon 
(1993) proposed a method for the simultaneous measurements of 27 
herbicides in the soil using HPLC. Kiang and Grob (1986) proposed a method 
for simultaneous measurement of 49 soil pollutants by capillary gas 
chromatography. 

Equipment and Products 

- Back and forth shaker. 

-Decanting funnels in borosilicate glass with ground Teflon stoppers: 
50, 125, 250 mL. 

- Columns for liquid chromatography, diameter: 1 cm, length: 50 cm, 
with a PTFE stopcock and a solvent tank. 

- 10 mL half cylindrical half conical volumetric tubes. 

- Rotary evaporator with 50, 125, 250 mL boiling flasks. 

- Regulated aluminium heating block and system of evaporation under 
flow of nitrogen (Fig. 13.2). Alternatively, a centrifugal evaporator 
under vacuum, type: speed-vac, can be used. 

- Gas phase chromatograph with several selective detectors (e.g. electron 
capture detector for halogenated compounds, thermoionic detector for 
organophosphorous compounds) and non-selective (e.g. flame ionization 
or mass spectrometry). The use of a glass needle injector is 
recommended, or failing that a split-splitless or on column injector 
(Pansuetal. 2001). 

- HPLC with UV detector if required; 

- Certified solvents, free from pesticides and pollutants. Each solvent 
should be tested by injection in the chromatograph after concentration 
by evaporation in the rotary evaporator (approximately 200 mL giving 
1 mL). The most commonly used solvents are acetone, petroleum ether, 
hexane, ethyl ether, methanol, acetonitrile, dichloromethane, ethyl 
acetate. 

- Different standard pesticides. 

- Activated florisil (60-80 mesh particle size, 3% of water). 

- Anhydrous sodium sulphate. 

- Sodium chloride. 



486 Mineralogical Analysis 

Extractions 

Acid Herbicides 

Jensen and Glass (1990) tested several techniques before recommending 
cold extraction with ethyl ether in acid medium. When well agitated for a 
rather long period, this technique is effective even for residues that have 
been incorporated in the soil for a year. A sufficient volume of water and 
acid have to be added to reduce the viscosity of the soil sample and ensure 
good contact with the ether phase. The authors advised three successive 
extractions with ether and one hour agitation each time. 

Triazine Herbicides 

Several different solvents can be used. For extractions in aqueous 
medium, a mixture of ethyl ether and petroleum ether (v/v) may be 
appropriate. For soils, we prefer a 1/5 mixture of methylene chloride and 
ethyl acetate. We recommend three successive extractions with a back 
and forth shaker on 20 g of sample with respectively, 100, 50 and 50 mL 
of solvent mixture. 

Organochlorides 

Wheeler and Thompson (1990) reviewed the large number of techniques 
used for the extraction of this type of compound. Methods used for soils 
are often based on those used in food analysis. A simple way is to agitate 
the soil sample (50 g) with acetone (100 mL) or an acetone-petroleum 
ether mixture on a back and forth shaker. With a wet sample, add 
anhydrous sodium sulphate as desiccant. 

Organophosphorous 

Freeze-drying is usually advised for the conservation of soil samples. 
Several solvents or mixtures of solvents have been used for standard extrac- 
tion of organophosphorous compounds in the soil: acetone, acetone-water, 
dichloromethane, ethyl acetate, acetone-hexane, acetone-dichloromethane, 
methanol-water (Barcelo and Lawrence, 1992). These compounds can be 
extracted jointly with organochlorides (acetone, acetone-hexane), pyre- 
thrinoids (acetone-hexane), and carbamates (acetone-dichloromethane). 

Carbamates 

Carbamates can be titrated with a rather complex technique of multi- 
residue analysis (Seiber 1990). The following method was tested by 
Pansu et al. (1981a) with an average extraction yield of 72 ± 8%. Prepare 
the soil samples by freeze-drying, sieve to 2 mm particle size and divide 
into sample specimens of 10 g each. Shake the sample specimen with 50 
mL methanol and 50 mL water for 6 h on a back-and-forth shaker, add 



Non-humic Molecules 487 

100 |LlL of 1 mol (HC1) L" 1 solution and filter under vacuum. Wash the 
residue with the extraction solution and add the washing solutions to the 
filtrate. Extract the filtrate three times in a 200 mL decanting funnel with 
respectively, 30, 20 and 20 mL chloroform. 

Pyrethrinoids 

In biological substrates, pyrethrinoids are extracted satisfactorily by two 
successive extractions with hexane in the presence of anhydrous sodium 
sulphate (Pansu et al. 1981b). For soils, a mixture of hexane with 3% 
acetone is more efficient. 

Preparation of the Extracts for Chromatography 

Principle 

Most extracts have to be purified before chromatography to eliminate 
interference with other lipidic compounds in the soil. The use of detectors 
that are selective for the main families of products (such as 
organochlorides or organo-phosphorus) makes it possible to limit the 
number of purifications. However, some of these detectors (electron 
capture for example) are very sensitive to pollution and the extracts need 
to be purified so as not to perturb the sensitivity of the detector. 

The extracts should be concentrated as much as possible to improve 
detection of ultra-trace residues. Extracts are usually concentrated in 
vacuum rotary evaporators during the first stage by micro-techniques 
such as gaseous nitrogen flow (Fig. 13.2) or a speed- vac centrifugal 
evaporator in the final stage. If too polar solvents are used for extraction 
(not easily eluted from chromatographic columns) or if they are 
incompatible with the detection system, the extract may have to be 
transferred into another solvent before injection. For example, if an 
electron capture detector is being used, chlorinated solvents have to be 
completely eliminated before injection. Except for volatile pesticides, this 
is accomplished by drying the extract several times, each time with 
dissolution in a non-halogenous solvent. 

Finally, some polar compounds require derivatization reactions before 
injection into the chromatograph. 

Acid Herbicides 

Purification can be achieved by separation into solvents (Jensen and Glass 
1990). The ether extracts (see "Acid Herbicides" in "Extractions" under 
Sect. 13.3.4) are agitated with a sodium bicarbonate aqueous solution. In 
the aqueous solution, acid herbicides are transformed into salts, while the 



488 Mineralogical Analysis 

majority of the coloured organic compounds remain in the organic phase. 
This phase is eliminated, and the acid herbicides are re-extracted with 
ether after acidification of the aqueous phase and saturation by sodium 
chloride. 

If gas chromatography is used for analysis, the extracts usually need to 
be methylated before injection, as acid herbicides are too polar. Several 
different reagents can be used in techniques similar to fatty acid 
methylation (cf. "Procedure" under Sect. 13.3.3 earlier). Diazomethane is 
a particularly effective methylation agent. 

Triazines 

The main lipids can be eliminated by acetonitrile-hexane partition. 
Concentrate the methylene chloride-ethyl acetate extracts to 2 mL (cf. 
"Triozine Herbicides"). Add 20 mL acetonitrile saturated with petroleum 
ether (AN). Add 10 mL petroleum ether saturated with acetonitrile (PE) 
and agitate. Recover the AN phase. Re-extract PE with 10 mL AN. 
Discard PE. Add all the AN phases in the decanting funnel, add 120 mL 
water and 10 mL of a NaCl saturated aqueous solution. Extract triazines 
twice with 50 mL of the ethyl ether/petroleum-ether v/v mixture. Mix the 
ether phases, dry on sodium sulphate and concentrate to the exact volume 
desired (2 mL for example) before injection. 

Organochlorides 

First the acetone extract must be transferred in a hexane phase. Put the 
acetone extract in a decanting funnel (cf. "Organochlorides" earlier in 
"Extractions" under Sect. 13.3.4) with four times its volume of water and 
25 mL hexane. Swirl gently and decant the organic phase. Extract the 
aqueous phase again with 25 mL hexane, mix the hexanic extracts, dry on 
anhydrous sodium sulphate and concentrate to 5 or 10 mL. 

Purify the extracts on a column filled with activated Florisil (3% 
water). Weigh 5 g of Florisil and place it in a column with a diameter of 1 
cm stopped with a glass wool plug and partially filled with petroleum 
ether. Add 2-3 cm of anhydrous sodium sulphate, rinse with 50 mL 
hexane until the liquid comes to the top the sodium sulphate phase. 

Deposit the hexanic extract on the column with a Pasteur pipette. 
Regulate the flow to approximately 15-20 drops per minute with the 
PTFE stopcock. When the level of the liquid comes to the top of the 
sodium sulphate phase, add a little hexane while rinsing with the pipette; 
repeat this procedure once. Elute with exactly the volume of hexane 
needed to obtain PCB, i.e. approximately 20-30 mL. This El eluate may 
also contain hexachlorobenzene (HCB) and dichlorodiphenylethane 
(DDE), one of the breakdown products of DDT. The exact quantity of 
solvent for the El eluate must first be determined under the same 



Non-humic Molecules 489 

conditions with standard mixtures. For PCB, these standards are made of 
sets of products providing typical chromatograms (e.g. dp5 and dp6 
mixtures). 

After collection of the El eluate, elution should be continued with the 
10% of dichloromethane in the petroleum ether (or hexane) mixture until a 
sufficient volume has been obtained for collection of the other organo- 
chlorinated compounds (E2 eluate). The exact volume of E2 should first be 
determined with one or more standard compounds, dieldrin for example. 

The E2 eluate has to be removed from the dichloromethane. Bring just to 
dry in a rotary evaporator. Add a little petroleum ether and bring just to dry 
again. Repeat this operation four or five times. Bring the El and E2 eluates 
to the exact volume of hexane chosen for chromatographic titration. 

Organophosphorous 

First the acetone extract has to be transferred in a hexanic phase. Place 
the extract in a decanting funnel (cf. "Organophosphorous" earlier in 
"Extractions" under Sec. 13.3.4) with four times its volume of water and 
25 mL methylene chloride. Swirl gently and decant the organic phase. 
Extract again with 25 mL methylene chloride, mix the extracts, dry them 
on anhydrous sodium sulphate and evaporate just to dry in a rotary 
evaporator. Add a little petroleum ether and bring just to dry again. 
Repeat this procedure three times. Rinse several times with 10 mL total 
volume of hexane at 10% of acetone, mix the rinsing solutions, stop and 
store for chromatographic determination. 

Carbamates 

Chloroformic extracts (cf. "Carbamates" earlier in "Extractions" under 
Sect. 13.3.4) can be purified in the following way. Evaporate to dry, 
recover in 10 mL methanol +10 mL 0.1 mol (HO) L~ solution, cool 15 
min, filter the suspended matter and wash the filter with a v/v mixture of 
water and 0.1 mol (HO) L _1 solution. Extract the filtrate three times with 
25 mL chloroform, dry the extracts on anhydrous sodium sulphate and 
transfer in a 100 mL round bottom boiling flask. To eliminate the 
chloroform, evaporate just to dry on the rotary evaporator, add a little 
methanol and again evaporate just to dry, repeat this procedure three 
times. Finally transfer in exactly 10 mL methanol in half cylindrical half 
conical volumetric tubes with screw caps, washing several times the 
boiling flask with a few mL of methanol. If the injection in the 
chromatograph cannot be carried out immediately, store at -20°C. To 
improve the detection limit the extract can be concentrated in the tubes 
under nitrogen flow at 40°C (Fig. 13.2). 



490 Mineralogical Analysis 

Note: Some carbamates can also be transformed into glycosides by 
plants. During extraction (See "Carbamates" in "Extractions" under Sect. 
13.3.4) these forms are not found in the chloroformic fraction, but remain 
in the hydro-alcoholic fraction. To identify them, it is necessary to break 
the glycoside bonds by reflux boiling saponification and to re-extract 
with chloroform (Pansu et al. 1981a). 

Pyrethrinoids 

The extracts of "Pyrethrinoids" in "Extractions" under Sec. 13.3.4 should 
be purified before being injected in a chromatograph equipped with an 
electron capture detector. Purification is carried out on chromatographic 
columns (diameter 1 cm) filled with 5 g Florisil at 3% of water and 2 g of 
anhydrous sodium sulphate. 

Two different procedures are used for extracts that contain fat 
substances and extracts that do not (Pansu et al. 1981b). In the absence of 
lipids, pyrethrinoids are recovered by elution with 70 mL of a mixture of 
10% ethyl ether in petroleum ether. In the presence of lipids, preliminary 
elution with approximately 110 mL hexane enables elimination of the 
lipids before pyrethrinoid elution as previously described. The exact 
volumes of the eluting solutions must first be determined by tests with 
standard pyrethrinoids (decamethrin) and by gravimetry of the eluted fat 
substances. Bring the eluate containing the pyrethrinoids just to dry and 
dissolve again in exactly 10 mL hexane in a half cylindrical half conical 
volumetric tube with a PTFE screw cap for chromatographic deter- 
mination. To improve detection, the extracts can subsequently be 
concentrated in the tubes. 

Chromatographic Determination 

A large range of columns and chromatographic conditions are suitable for 
the determination of pesticides and pollutants, among which the techni- 
ques later are worth mentioning. 

Acid Herbicides 

The measurement of acid herbicides by GPC requires prelimin-ary 
methylation of the acid functions (cf. "Fractionation of Fatty Acids and 
Unsaponifiable" earlier). For the separation of methylated esters of 2,4D 
and 2,4, 5T, use a column 1.5 m in length with an interior diameter of 4 
mm filled with a 5% OV225 phase on a chromosorbW support of 100- 
120 mesh particle size at an isothermal temperature of 220°C, with a 
electron capture detector. Acid herbicides can also be measured by liquid 
chromatography. 



Non-humic Molecules 491 

Triazines 

Atrazine and simazine can be measured by GPC under similar conditions 
to those described earlier for acid herbicides but using a thermionic 
detector. For simultaneous measurement of all nitrogenized herbicides, 
it is preferable to use a capillary column such as PTE5 30 m x 0.25 mm 
ID (Supelco), with a temperature programme of 40°C (5 min) to 100°C 
(30°C min" 1 ) then to 275°C (5°C min" 1 ). 

Organ